Pyrido[3,4-d]pyrimidine derivative and pharmaceutically acceptable salt thereof

ABSTRACT

The purpose of the present invention is to provide a compound having an excellent CDK4/6 inhibiting activity. The present invention is a compound represented by general formula (I) or a pharmaceutically acceptable salt thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a National Stage of International Application No. PCT/JP2016/065770 filed May 27, 2016, claiming priority based on Japanese Patent Application No. 2015-110684 filed May 29, 2015, the contents of all of which are incorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention relates to a pyrido[3,4-d]pyrimidine derivative and a pharmaceutically acceptable salt thereof. In particular, the present invention relates to a compound that exhibits an inhibitory activity against cyclin-dependent kinase 4 and/or cyclin-dependent kinase 6 (hereinafter referred to as “CDK4/6”) and that is useful for the prevention or treatment of rheumatoid arthritis, arteriosclerosis, pulmonary fibrosis, cerebral infarction, or cancer.

BACKGROUND ART

Cell growth, which is a process involving proliferation and division of cells, occurs in response to various stimuli.

Pathological conditions caused by hyperproliferation of cells, such as cancer, are characterized by uncontrollable cell cycle progression and thus excessive progression of the cell cycle, for example, resulting from abnormality in genes or proteins that directly or indirectly regulate the cell cycle progression. Substances that regulate hyperproliferation of cells through control of the cell cycle can be used for the treatment of various pathological conditions characterized by uncontrollable or unwanted cell growth.

Cell cycle progression is a complicated process involving highly regulated transition of phases and multiple checkpoints.

Cyclin-dependent kinases and associated serine/threonine protein kinases are important intracellular enzymes that play essential roles in the regulation of division and proliferation of cells. Catalytic subunits of cyclin-dependent kinases are activated by regulatory subunits known as cyclins, and multiple cyclins have been identified in mammals (NPL 1).

The retinoblastoma (Rb) protein is a checkpoint protein for transition from the G1 phase to the S phase in the cell cycle. The Rb protein associates with the E2F transcription factor family and inhibits the activity thereof in the absence of appropriate growth stimulation (NPLs 2 and 3). A cell stimulated by a mitogen enters the S phase through synthesis of cyclin D, which is a CDK4/6 activator. The cyclin D-bound CDK 4/6 inactivates the Rb protein through phosphorylation. The phosphorylation of the Rb protein releases E2F in order to indirective the transcription of a gene necessary for the S phase. The complete inactivation of the Rb protein requires phosphorylation of both cyclin D-CDK4/6 and cyclin E-CDK2. The phosphorylation of the Rb protein by CDK4/6 at a specific site is essential in the phosphorylation of cyclin E-CDK2 (NPL 4). Thus, cyclin D-CDK4/6 is an important enzyme complex which controls the transition from the G1 phase to the S phase.

CDK2 forms a complex with cyclin E and also forms a complex with cyclin A. CDK2 also acts on steps subsequent to the S phase and is responsible for DNA replication. The inhibition of CDK2 probably leads to the expression of genotoxicity (NPL 5).

Cyclin D has a molecular mechanism that positively regulates the activity of CDK4/6. In contrast, p16 encoded by the INK4a gene negatively regulates the activity of CDK4/6 (NPL 6).

CDK inhibitors can be used for the treatment of various diseases caused by abnormal cell growth, such as cancer, cardiovascular disorder, renal disease, specific infections, and autoimmune diseases. CDK inhibitors is also expected to be effective for the treatment of diseases including but not limited to rheumatoid arthritis, arteriosclerosis, pulmonary fibrosis, cerebral infarction, and cancer. The inhibition of cell cycle progression and cell growth through CDK inhibition is expected to be effective for such a disease on the basis of the technical findings described below.

Rheumatoid arthritis involves the formation of pannus through hyperproliferation of synovial cells. This hyperproliferation can be reduced by the introduction of p16 into an affected area of a model animal or the administration of a CDK4/6 inhibitor to the animal (NPLs 7 to 9). A CDK4-cyclin D complex regulates the production of MMP3 in synovial cells derived from a patient with rheumatoid arthritis. The negative regulation of the activity of CDK4/6 inhibits not only the proliferation but also production of MMP3 (NPL 10).

Thus, CDK4/6 inhibitors are expected to exhibit both an inhibitory effect on proliferation of synovial cells and a cartilage protective effect in rheumatoid arthritis.

A pathway for the regulation of cell growth including genes responsible for the checkpoints in the G1 and S phases of the cell cycle is associated with plaque progression, stenosis, and restenosis after angiogenesis. The overexpression of the CDK inhibitory protein p21 inhibits angiogenesis and subsequent growth of vascular smooth muscle and intimal hyperplasia (NPLs 11 and 12).

Abnormal regulation of the cell cycle is also associated with polycystic kidney disease, which is characterized by growth of cysts filled with fluid in the renal tubule. A small-molecule CDK inhibitor is effective for the treatment of the disease (NPL 13).

The induction of expression of the cell cycle inhibitory protein p21 with an adenoviral vector is effective in a murine pulmonary fibrosis model (NPL 14).

The level of cyclin D1/CDK4 is known to increase in a rat cerebral infarction model in association with neuronal death caused by local ischemia. The neuronal death is reduced by administering flavopiridol, which is a nonselective CDK inhibitor (NPL 15).

The cyclin D-CDK4/6-INK4a-Rb pathway is frequently detected in human cancer caused by abnormality of any factors contributing to growth of cancer cells, such as loss of functional p16INK4a, overexpression of cyclin D1, overexpression of CDK4, or loss of functional Rb (NPLs 16 to 18). Such abnormality promotes the cell cycle progression from the G1 phase to the S phase, and this pathway certainly plays an important role in oncogenic transformation or abnormal growth of cancer cells.

CDK4/6 inhibitors may be effective, particularly for tumors involving abnormality in genes that activate the CDK4/6 kinase activity, such as cancers involving the translocation of cyclin D, cancers involving the amplification of cyclin D, cancers involving the amplification or overexpression of CDK4 or CDK6, and cancers involving the inactivation of p16. CDK4/6 inhibitors may be effective for the treatment of cancers involving genetic abnormality in the upstream regulator of cyclin D, the amount of which increases due to defects in the upstream regulator.

In fact, many compounds that inhibit the CDK4/6 activity have been synthesized and disclosed in the art, and such compounds have been clinically tested for the treatment of cancers, such as breast cancer (NPL 19).

Most acute and severe radiotherapeutic and chemotherapeutic toxicities are caused by the effects on stem cells and progenitor cells. A CDK4/6 inhibitor causes temporary cell cycle arrest to hematopoietic stem and progenitor cells, and protects them from radiotherapeutic or chemotherapeutic cytotoxicity. After the treatment with the inhibitor, hematopoietic stem and progenitor cells (HSPCs) return from the temporary dormancy and then function normally. Thus, the chemotherapeutic resistance with use of a CDK4/6 inhibitor is expected to provide a significant protection of bone marrow (NPL 20).

Hence, CDK4/6 inhibitors are expected to be effective for the treatment of rheumatoid arthritis, arteriosclerosis, pulmonary fibrosis, cerebral infarction, or cancer, and the protection of bone marrow, in particular, for the treatment of rheumatoid arthritis or cancer and the protection of bone marrow.

PTL 1 and NPL 21 disclose CDK4 inhibitors, PTLs 2 and 3 and NPLs 22 to 24 disclose CDK4/6-containing CDK inhibitors, and NPL 25 discloses CDK4/FLT3 inhibitors.

Pyrido[3,4-d]pyrimidine derivatives exhibit an inhibitory effect on Mps1 (also known as TTK) (PTL 4). This inhibitory effect is completely different from the CDK4/6 inhibitory effect disclosed in the present invention.

NPL 26 and NPL 27 disclose that a plurality of pyrido[3,4-d]pyrimidine derivatives exhibit a CDK2 inhibitory activity, which is completely different from the superior CDK4/6 inhibitory effect exhibited by the present invention.

LIST OF CITATIONS Patent Literature

-   [PTL 1] WO2003/062236 -   [PTL 2] WO2010/020675 -   [PTL 3] WO2010/075074 -   [PTL 4] WO2014/037750

Non-Patent Literature

-   [NPL 1] Johnson D. G. and Walker C. L., Annual Review of     Pharmacology and Toxicology 1999; 39: p. 295-312 -   [NPL 2] Ortega et al., Biochimica et Biophysica Acta-Reviews on     Cancer 2002; 1602 (1): p. 73-87 -   [NPL 3] Shapiro, Journal of Clinical Oncology 2006; 24 (11): p.     1770-1783 -   [NPL 4] Lundberg et al., Molecular and Cellular Biology 1998; 18     (2): p. 753-761 -   [NPL 5] Andrew J. Olaharski, PLoS Computational Biology 2009; 5 (7):     e1000446 -   [NPL 6] Kamb et al., Science 1994; 264 (5157): p. 436-440 -   [NPL 7] Taniguchi, K et al., Nature Medicine, Vol. 5, p. 760-767     (1999) -   [NPL 8] Sekine, C et al., Journal of immunology 2008, 180: p.     1954-1961 -   [NPL 9] Hosoya, T et al., Annnl Rheumatic Diseases 2014 Aug. 27 Epub     ahead of print -   [NPL 10] Nonomura Y et al., Arthritis & Rheumatology 2006, July; 54     (7): p. 2074-83 -   [NPL 11] Chang M. W. et al., Journal of Clinical Investigation,     1995, 96: p. 2260 -   [NPL 12] Yang Z-Y. et al., Proceedings of the National Academy of     Sciences (USA) 1996, 93: p. 9905 -   [NPL 13] Bukanov N. O. et al., Nature, 2006, 4444: p. 949-952 -   [NPL 14] American Journal Physiology: Lung Cellular and Molecular     Physiology, 2004, Vol. 286, p. L727-L733 -   [NPL 15] Proceedings of the National Academy of Sciences of the     United States of America, 2000, Vol. 97, p. 10254-10259 -   [NPL 16] Science, Vol. 254, p. 1138-1146 (1991) -   [NPL 17] Cancer Research, 1993, Vol. 53, p. 5535-5541 -   [NPL 18] Current Opinion in Cell Biology, 1996, Vol. 8, p. 805-814 -   [NPL 19] Guha M, Nature Biotechnology 2013, March; 31 (3): p. 187 -   [NPL 20] Journal of Clinical Investigation 2010; 120 (7): p.     2528-2536 Soren M. Johnson -   [NPL 21] Journal of Medicinal Chemistry, 2005, 48, p. 2371-2387 -   [NPL 22] Journal of Medicinal Chemistry, 2000, 43, p. 4606-4616 -   [NPL 23] Journal of Medicinal Chemistry, 2005, 48, p. 2388-2406 -   [NPL 24] Journal of Medicinal Chemistry, 2010, 53, p. 7938-7957 -   [NPL 25] Journal of Medicinal Chemistry, 2014, 57, p. 3430-3449 -   [NPL 26] Organic & Biomolecular Chemistry, 2015, 13, p. 893-904 -   [NPL 27] Rapid Discovery of Pyrido[3,4-d]pyrimidine Inhibitors of     Monopolar Spindle Kinase 1 (MPS1) Using a Structure-Based     Hybridization Approach, Paolo Innocenti et al, J. Med. Chem.,     Article ASAP, Publication Date (Web): Apr. 7, 2016, DOI:     10.1021/acs.jmedchem.5b01811.

SUMMARY OF INVENTION Problem to be Solved by the Invention

An object of the present invention is to provide a compound exhibiting a superior CDK4/6 inhibitory activity.

Means to Solve the Problem

The present inventors have conducted extensive studies for solving the problems described above and have found that a novel pyrido[3,4-d]pyrimidine derivative represented by Formula (I) exhibits a CDK4/6 inhibitory activity. The present invention has been accomplished on the basis of this finding.

The present invention includes the following aspects:

Aspect (1): A compound represented by Formula (I), or a pharmaceutically acceptable salt thereof:

[wherein L represents —NR⁵—, —O—, or —S—; R⁵ represents a hydrogen atom or a C₁₋₆ alkyl group substituted with zero to two —OH groups, zero to two C₁₋₈ alkoxy groups, and zero to six fluorine atoms; R¹ represents a C₁₋₈ alkyl, C₃₋₁₂ cycloalkyl, (C₃₋₁₂ cycloalkyl)-C₁₋₆ alkyl, 4- to 12-membered heterocyclyl, (4- to 12-membered heterocyclyl)-C₁₋₆ alkyl, C₆₋₁₀ aryl, (C₆₋₁₀ aryl)-C₁₋₆ alkyl, 5- to 10-membered heteroaryl, (5- to 10-membered heteroaryl)-C₁₋₆ alkyl, C₁₋₈ alkylsulfonyl, or C₁₋₈ acyl group; each of the heteroatom-containing groups represented by R¹ contains one to four heteroatoms selected from oxygen, sulfur, and nitrogen atoms; R¹ is optionally substituted with one to six substituents selected from the group consisting of a halogen atom, ═O, —OH, —CN, —COOH, —COOR⁶, —R⁷, a C₃₋₆ cycloalkyl group substituted with zero to two —OH groups, zero to two C₁₋₈ alkoxy groups, and zero to six fluorine atoms, a 3- to 10-membered heterocyclyl group substituted with zero to two —OH groups, zero to two C₁₋₈ alkoxy groups, and zero to six fluorine atoms, a C₁₋₈ acyl group substituted with zero to two —OH groups, zero to two C₁₋₈ alkoxy groups, and zero to six fluorine atoms, and a C₁₋₈ alkoxy group substituted with zero to two —OH groups, zero to two C₁₋₈ alkoxy groups, and zero to six fluorine atoms; R⁶ and R⁷ each independently represent a C₁₋₆ alkyl group substituted with zero to two —OH groups, zero to two C₁₋₈ alkoxy groups, and zero to six fluorine atoms; R² represents a C₁₋₈ alkyl, C₃₋₈ cycloalkyl, 4- to 6-membered heterocyclyl, or C₁₋₈ acyl group, —COOR⁸, or —CONR⁹R¹⁰; each of the C₁₋₈ alkyl and C₃₋₈ cycloalkyl groups represented by R² is substituted with zero or one —OH group, zero to two C₁₋₈ alkoxy groups substituted with zero or one —OH group, zero or one C₁₋₄ alkoxy group, and zero to three fluorine atoms, and zero to five fluorine atoms; R² is neither an unsubstituted C₁₋₈ alkyl, nor unsubstituted C₃₋₈ cycloalkyl, nor trifluoromethyl group; R⁸, R⁹, and R¹⁰ each independently represent a hydrogen atom or a C₁₋₈ alkyl group; the 4- to 6-membered heterocyclyl group represented by R² is optionally substituted with one to four substituents selected from the group consisting of a fluorine atom, —OH, and C₁₋₄ alkyl and C₁₋₄ alkoxy groups; each of the C₁₋₈ acyl group, —COOR⁸, and —CONR⁹R¹⁰ represented by R² is optionally substituted with one to four substituents selected from the group consisting of a fluorine atom, —OH, and a C₁₋₄ alkoxy group; R⁹ and R¹⁰ of —CONR⁹R¹⁰ represented by R² are optionally bonded via a single bond or —O— to form a ring including the nitrogen atom bonded to R⁹ and R¹⁰; the heterocyclyl group represented by R² having a 4- or 5-membered ring contains one oxygen heteroatom, and the heterocyclyl group having a 6-membered ring contains one or two oxygen heteroatoms; R³ represents a hydrogen atom, a C₁₋₈ alkyl group, or a halogen atom; X represents CR¹¹ or a nitrogen atom; Y represents CR¹² or a nitrogen atom; Z represents CR¹³ or a nitrogen atom; R¹¹ to R¹³ each independently represent a hydrogen, fluorine, chlorine atom, a C₁₋₆ alkyl, or C₁₋₆ alkoxy group; R⁴ represents -A¹-A²-A³; A¹ represents a single bond, a C₁₋₈ alkylene, C₂₋₈ alkenylene, or C₂₋₈ alkynylene group; one or two sp³ carbon atoms at any positions of A¹ are optionally replaced with one or two structures selected from the group consisting of —O—, —NR¹⁴—, —C(═O)—, —C(═O)—O—, —O—C(═O)—, —O—C(═O)—O—, —C(═O)—NR¹⁵—, —O—C(═O)—NR¹⁶—, —NR¹⁷—C(═O)—, —NR¹⁸—C(═O)—O—, —NR¹⁹—C(═O)—NR²⁰—, —S(═O)_(p)—, —S(═O)₂—NR²¹—, —NR²²—S(═O)₂—, and —NR²³—S(═O)₂—NR²⁴—, and a structure of —O—O—, —O—NR¹⁴—, —NR¹⁴—O—, —O—CH₂—O—, —O—CH₂—NR¹⁴—, or —NR¹⁴—CH₂—O— is not formed in the case of replacement of two sp³ carbon atoms; A² represents a single bond, a C₁₋₇ alkylene, C₃₋₁₂ cycloalkylene, C₃₋₁₂ cycloalkylidene, 4- to 12-membered heterocyclylene, 4- to 12-membered heterocyclylidene, C₆₋₁₀ arylene, or 5- to 10-membered heteroarylene group; A³ represents a halogen atom, —CN, —NO₂, —R²⁵, —OR²⁶, —NR²⁷R²⁸, —C(═O)R²⁹, —C(═O)—OR³⁰, —O—C(═O)R³¹, —O—C(═O)—NR³²R³³, —C(═O)—NR³⁴R³⁵, —NR³⁶—C(═O)R³⁷, —NR³⁸—C(═O)—OR³⁹, —S(═O)₂—R⁴, —S(═O)₂—NR⁴¹R⁴², or —NR⁴³—S(═O)₂R⁴⁴; A³ represents —R²⁵, if the A¹ end on the A² side has a structure selected from the group consisting of —O—, —NR¹⁴—, —C(═O)—, —C(═O)—O—, —O—C(═O)—, —O—C(═O)—O—, —C(═O)—NR¹⁵—, —O—C(═O)—NR¹⁶—, —NR¹⁷—C(═O)—, —NR¹⁸—C(═O)—O—, —NR¹⁹—C(═O)—NR²⁰—, —S(═O)—, —S(═O)₂—NR²¹—, —NR²²—S(═O)₂—, and —NR²³—S(═O)₂—NR²⁴— and A² is a single bond; R¹⁴, R³², R³⁴, R³⁶, R³⁸, R⁴¹, and R⁴³ each independently represent a hydrogen atom, a C₁₋₈ alkyl, C₁₋₈ acyl, C₁₋₈ alkylsulfonyl, 4- to 12-membered heterocyclyl, C₃₋₁₂ cycloalkyl, C₆₋₁₀ aryl, 5- to 10-membered heteroaryl, (4- to 12-membered heterocyclyl)-C₁₋₃ alkyl, (C₃₋₁₂ cycloalkyl)-C₁₋₃ alkyl, (C₆₋₁₀ aryl)-C₁₋₃ alkyl, or (5- to 10-membered heteroaryl)-C₁₋₃ alkyl group; R¹⁵ to R³¹, R³³, R³⁵, R³⁷, R³⁹, R⁴⁰, R⁴², and R⁴⁴ each independently represent a hydrogen atom or a C₁₋₈ alkyl, 4- to 12-membered heterocyclyl, C₃₋₁₂ cycloalkyl, C₆₋₁₀ aryl, 5- to 10-membered heteroaryl, (4- to 12-membered heterocyclyl)-C₁₋₃ alkyl, (C₃₋₁₂ cycloalkyl)-C₁₋₃ alkyl, (C₆₋₁₀ aryl)-C₁₋₃ alkyl, or (5- to 10-membered heteroaryl)-C₁₋₃ alkyl group; A¹, A², A³, and R¹⁴ to R⁴⁴ in A¹, A², and A³ are each optionally substituted with one to four substituents selected from the group consisting of —OH, ═O, —COOH, —SO₃H, —PO₃H, —CN, —NO₂, a halogen atom, a C₁₋₈ alkyl group substituted with zero to two —OH groups, zero to two —OR⁴⁵ groups, and zero to six fluorine atoms, a C₃₋₁₂ cycloalkyl group substituted with zero to two —OH groups, zero to two —OR⁴⁶ groups, and zero to six fluorine atoms, a C₁₋₈ alkoxy group substituted with zero to two —OH groups, zero to two —OR⁴⁷ groups, and zero to six fluorine atoms, and a 4- to 12-membered heterocyclyl group substituted with zero to two —OH groups, zero to two —OR⁴⁹ groups, and zero to six fluorine atoms; R¹⁴ to R⁴⁴ are optionally bonded in A¹, A², or A³ or between A¹ and A², between A¹ and A³, or between A² and A³ via a single bond, —O—, —NR⁵⁰—, or —S(═O)_(p)— to form a ring; R¹¹ or R¹³ is optionally bonded to A¹, A², or A³ via a single bond, —O—, —NR⁵¹—, or —S(═O)_(p)— to form a ring; R⁴⁵ to R⁵¹ each represent a hydrogen atom or a C₁₋₄ alkyl group substituted with zero or one —OH group and zero to six fluorine atoms; p represents an integer of 0 to 2; and each of the heteroatom-containing groups represented by A¹, A², and A³ contains one to four heteroatoms selected from oxygen, sulfur, and nitrogen atoms]. Aspect (2): The compound or pharmaceutically acceptable salt thereof according to Aspect (1), wherein L represents —NH—. Aspect (3): The compound or pharmaceutically acceptable salt thereof according to Aspect (1) or (2), wherein R1 represents a C1-8 alkyl, C3-12 cycloalkyl, (C3-12 cycloalkyl)-C1-6 alkyl, 4- to 12-membered heterocyclyl, or (4- to 12-membered heterocyclyl)-C1-6 alkyl group. Aspect (4): The compound or pharmaceutically acceptable salt thereof according to any one of Aspects (1) to (3), wherein R2 is a C1-8 alkyl group substituted with one to four fluorine atoms. Aspect (5): The compound or pharmaceutically acceptable salt thereof according to any one of Aspects (1) to (3), wherein R2 is a C1-8 alkyl group substituted with zero or one —OH group and zero to two C1-8 alkoxy groups substituted with zero or one —OH group, zero or one C1-4 alkoxy group, and zero to three fluorine atoms. Aspect (6): The compound or pharmaceutically acceptable salt thereof according to any one of Aspects (1) to (3), wherein R2 is a 4- to 6-membered heterocyclyl group optionally substituted with one to four substituents selected from the group consisting of a fluorine atom, —OH, and C1-4 alkyl and C1-4 alkoxy groups. Aspect (7): The compound or pharmaceutically acceptable salt thereof according to any one of Aspects (1) to (3), wherein R2 is —COOR8, —CONR9R10, or a C1-8 acyl group, optionally each group being substituted with one to four substituents selected from the group consisting of a fluorine atom, —OH, and a C1-8 alkoxy group. Aspect (8): The compound or pharmaceutically acceptable salt thereof according to any one of Aspects (1) to (7), wherein X represents CR11, Y represents CR12, and Z represents CR13. Aspect (9): The compound or pharmaceutically acceptable salt thereof according to any one of Aspects (1) to (7), wherein X represents a nitrogen atom, Y represents CR12, and Z represents CR13. Aspect (10): The compound or pharmaceutically acceptable salt thereof according to any one of Aspects (1) to (7), wherein X represents CR11, Y represents a nitrogen atom, and Z represents CR13. Aspect (11): The compound or pharmaceutically acceptable salt thereof according to any one of Aspects (1) to (7), wherein X represents CR11, Y represents CR12, and Z represents a nitrogen atom. Aspect (12): The compound or pharmaceutically acceptable salt thereof according to any one of Aspects (1) to (11), wherein A1 is a single bond. Aspect (13): The compound or pharmaceutically acceptable salt thereof according to any one of Aspects (1) to (11), wherein A1 represents a C1-8 alkylene group, and no sp3 carbon atom in A1 is replaced with another structure. Aspect (14): The compound or pharmaceutically acceptable salt thereof according to any one of Aspects (1) to (11), wherein A1 represents a C1-8 alkylene group, and one sp3 carbon atom at any position of A1 is replaced with —O—. Aspect (15): The compound or pharmaceutically acceptable salt thereof according to any one of Aspects (1) to (11), wherein A1 represents a C1-8 alkylene group, and one sp3 carbon atom at any position of A1 is replaced with —NR14-. Aspect (16): The compound or pharmaceutically acceptable salt thereof according to any one of Aspects (1) to (11), wherein A1 represents a C1-8 alkylene group, one sp3 carbon atom at any position of A1 is replaced with —NR14-, and one sp3 carbon atom at any other position of A1 is optionally replaced with —O—. Aspect (17): The compound or pharmaceutically acceptable salt thereof according to any one of Aspects (1) to (16), wherein A2 represents a 4- to 12-membered heterocyclylene group; and A2 is optionally substituted with one to four substituents selected from the group consisting of —OH, —COOH, —SO3H, —PO3H, —CN, —NO2, a halogen atom, a C1-8 alkyl group optionally substituted with zero to two —OH groups, zero to two —OR45 groups, and zero to six fluorine atoms, a C3-12 cycloalkyl group optionally substituted with zero to two —OH groups, zero to two —OR46 groups, and zero to six fluorine atoms, a C1-8 alkoxy group optionally substituted with zero to two —OH groups, zero to two —OR47 groups, and zero to six fluorine atoms, and a 4- to 12-membered heterocyclyl group substituted with zero to two —OH groups, zero to two —OR49 groups, and zero to six fluorine atoms. Aspect (18): The compound or pharmaceutically acceptable salt thereof according to any one of Aspects (1) to (16), wherein A2 represents a 4- to 12-membered heterocyclylene group substituted with ═O; and A2 is optionally substituted with one to four substituents selected from the group consisting of —OH, ═O, —COOH, —SO3H, —PO3H, —CN, —NO2, a halogen atom, a C1-8 alkyl group substituted with zero to two —OH groups, zero to two —OR45 groups, and zero to six fluorine atoms, a C3-12 cycloalkyl group substituted with zero to two —OH groups, zero to two —OR46 groups, and zero to six fluorine atoms, a C1-8 alkoxy group substituted with zero to two —OH groups, zero to two —OR47 groups, and zero to six fluorine atoms, and a 4- to 12-membered heterocyclyl group substituted with zero to two —OH groups, zero to two —OR49 groups, and zero to six fluorine atoms. Aspect (19): The compound or pharmaceutically acceptable salt thereof according to any one of Aspects (1) to (18), wherein X represents CR11, Y represents CR12, Z represents CR13, and R11 or R13 is bonded to A1, A2, or A3 via a single bond, —O—, —NR51—, or —S(═O)p- to form a ring. Aspect (20): The compound or pharmaceutically acceptable salt thereof according to any one of Aspects (1) to (19), wherein A3 is a hydrogen atom. Aspect (21): The compound or pharmaceutically acceptable salt thereof according to any one of Aspects (1) to (19), wherein A3 is a halogen atom, —CN, —R25, —OR26, —NR27R28, —C(═O)R29, or —C(═O)—OR30, and R25 to R30 each independently represent a hydrogen atom, an optionally substituted C1-8 alkyl group, an optionally substituted 4- to 12-membered heterocyclyl group, an optionally substituted C3-12 cycloalkyl group, an optionally substituted (4- to 12-membered heterocyclyl)-C1-3 alkyl group, or an optionally substituted (C3-12 cycloalkyl)-C1-3 alkyl group. Aspect (22): The compound or pharmaceutically acceptable salt thereof according to any one of Aspects (1) to (21), wherein R3 is a hydrogen atom. Aspect (23): The compound or pharmaceutically acceptable salt thereof according to any one of Aspects (1) to (21), wherein R3 represents a C1-4 alkyl group, a fluorine atom, or a chlorine atom. Aspect (24): The compound, or pharmaceutically acceptable salt thereof, selected from;

-   6-(difluoromethyl)-N8-isopropyl-N2-(5-piperazin-1-yl-2-pyridyl)pyrido[3,4-d]pyrimidine-2,8-diamine -   (1R)-1-[8-(isopropylamino)-2-[(5-piperazin-1-yl-2-pyridyl)amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol -   1-[2-[(5-piperazin-1-yl-2-pyridyl)amino]-8-(tetrahydrofuran-3-ylamino)pyrido[3,4-d]pyrimidin-6-yl]ethanol -   1-[2-[(5-piperazin-1-yl-2-pyridyl)amino]-8-(tetrahydropyran-3-ylamino)pyrido[3,4-d]pyrimidin-6-yl]ethanol -   N8-isopropyl-6-[(1R)-1-methoxyethyl]-N2-(6-piperazin-1-ylpyridazin-3-yl)pyrido[3,4-d]pyrimidine-2,8-diamine -   N8-isopropyl-6-[(1R)-1-methoxyethyl]-N2-[5-(piperazin-1-ylmethyl)-2-pyridyl]pyrido[3,4-d]pyrimidine-2,8-diamine -   1-[6-[[6-[(1R)-1-hydroxyethyl]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]piperazin-2-one -   1-[6-[[5-chloro-6-[(1R)-1-hydroxyethyl]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]piperazin-2-one -   (1R)-1-[2-[(6-piperazin-1-ylpyridazin-3-yl)amino]-8-(tetrahydropyran-4-ylamino)pyrido[3,4-d]pyrimidin-6-yl]ethanol -   (1R)-1-[2-[(6-piperazin-1-ylpyridazin-3-yl)amino]-8-[[(3S)-tetrahydropyran-3-yl]amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol -   (1R)-1-[2-[(6-piperazin-1-ylpyridazin-3-yl)amino]-8-[[(3R)-tetrahydropyran-3-yl]amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol -   (1R)-1-[2-[[5-(piperazin-1-ylmethyl)-2-pyridyl]amino]-8-(tetrahydropyran-4-ylamino)pyrido[3,4-d]pyrimidin-6-yl]ethanol -   (1R)-1-[2-[[5-(piperazin-1-ylmethyl)-2-pyridyl]amino]-8-[[(3S)-tetrahydropyran-3-yl]amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol -   (1R)-1-[2-[[5-(piperazin-1-ylmethyl)-2-pyridyl]amino]-8-[[(3R)-tetrahydropyran-3-yl]amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol -   1-[6-[[6-[(1R)-1-hydroxyethyl]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-2-yl]amino]pyridazin-3-yl]piperidin-4-ol -   (1R)-1-[8-(isopropylamino)-2-[(6-piperazin-1-ylpyridazin-3-yl)amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol -   1-[[6-[[6-[(1R)-1-hydroxyethyl]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]methyl]piperazin-2-one -   6-[(1R)-1-methoxyethyl]-N2-[5-(piperazin-1-ylmethyl)-2-pyridyl]-N8-[(3S)-tetrahydropyran-3-yl]pyrido[3,4-d]pyrimidine-2,8-diamine -   6-[(1R)-1-methoxyethyl]-N2-(6-piperazin-1-ylpyridazin-3-yl)-N8-[(3S)-tetrahydropyran-3-yl]pyrido[3,4-d]pyrimidine-2,8-diamine -   6-[(1R)-1-methoxyethyl]-N2-[5-(piperazin-1-ylmethyl)-2-pyridyl]-N8-(tetrahydropyran-4-ylmethyl)pyrido[3,4-d]pyrimidine-2,8-diamine -   N8-isopropyl-6-[(1R)-1-methoxyethyl]-N2-(5-piperazin-1-ylpyrazin-2-yl)pyrido[3,4-d]pyrimidine-2,8-diamine -   N8-isopropyl-6-[(1R)-1-methoxyethyl]-N2-[6-[(2S)-2-methylpiperazin-1-yl]pyridazin-3-yl]pyrido[3,4-d]pyrimidine-2,8-diamine -   N8-isopropyl-6-[(1R)-1-methoxyethyl]-N2-[6-[(2R)-2-methylpiperazin-1-yl]pyridazin-3-yl]pyrido[3,4-d]pyrimidine-2,8-diamine -   (1R)-1-[2-[[6-(4,7-diazaspiro[2.5]octan-7-yl)pyridazin-3-yl]amino]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-6-yl]ethanol -   (1R)-1-[2-[[5-(4,7-diazaspiro[2.5]octan-7-ylmethyl)-2-pyridyl]amino]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-6-yl]ethanol -   2-[1-[[6-[[6-[(1R)-1-hydroxyethyl]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]methyl]-4-piperidyl]propan-2-ol -   (1R)-1-[2-[[5-[[4-(2-hydroxyethyl)piperazin-1-yl]methyl]-2-pyridyl]amino]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-6-yl]ethanol -   (1R)-1-[2-[[5-[2-(dimethylamino)ethoxy]-2-pyridyl]amino]-8-[[(3S)-tetrahydropyran-3-yl]amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol -   (1R)-1-[2-[[6-(4-methylpiperazin-1-yl)pyridazin-3-yl]amino]-8-[[(3S)-tetrahydropyran-3-yl]amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol -   2-hydroxy-1-[4-[6-[[6-[(1R)-1-hydroxyethyl]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-2-yl]amino]pyridazin-3-yl]piperazin-1-yl]ethanone -   1-[6-[[8-(isopropylamino)-6-[(2S)-tetrahydrofuran-2-yl]pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]piperazin-2-one -   (1R)-1-[8-(isopropylamino)-2-(5,6,7,8-tetrahydro-1,6-naphthyridin-2-ylamino)pyrido[3,4-d]pyrimidin-6-yl]ethanol -   2-[4-[[6-[[6-[(1R)-1-hydroxyethyl]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]methyl]piperazin-1-yl]-2-methyl-propan-1-ol -   4-[6-[[6-[(1R)-1-hydroxyethyl]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]-1-[(2S)-2-hydroxypropyl]-1,4-diazepan-5-one -   4-[6-[[6-[(1R)-1-hydroxyethyl]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]-1-[(2R)-2-hydroxypropyl]-1,4-diazepan-5-one -   N8-isopropyl-N2-[5-(piperazin-1-ylmethyl)-2-pyridyl]-6-[(2S)-tetrahydrofuran-2-yl]pyrido[3,4-d]pyrimidine-2,8-diamine -   1-[6-[[6-[(1R)-1-hydroxyethyl]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-2-yl]amino]-2-methyl-3-pyridyl]piperazin-2-one -   1-[6-[[8-(isopropylamino)-6-[(3S)-tetrahydrofuran-3-yl]pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]piperazin-2-one -   (1R)-1-[2-(5,6,7,8-tetrahydro-1,6-naphthyridin-2-ylamino)-8-[[(3S)-tetrahydropyran-3-yl]amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol -   1-[6-[[8-(isopropylamino)-6-(3-methyloxetan-3-yl)pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]piperazin-2-one -   (1R)-1-[2-[[5-[4-(dimethylamino)cyclohexoxy]-2-pyridyl]amino]-8-[[(3S)-tetrahydropyran-3-yl]amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol -   6-[(1R)-1-methoxyethyl]-N2-[5-(piperazin-1-ylmethyl)-2-pyridyl]-N8-propyl-pyrido[3,4-d]pyrimidine-2,8-diamine -   6-[(1R)-1-methoxyethyl]-N2-(6-piperazin-1-ylpyridazin-3-yl)-N8-propyl-pyrido[3,4-d]pyrimidine-2,8-diamine -   1-[[6-[[6-(difluoromethyl)-8-[(4-methylcyclohexyl)amino]pyrido[6-[[6-hexy)amino]pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]methyl]piperidine-4-carboxylic     acid -   (1R)-1-[8-(ethylamino)-2-[[5-[[4-(2-hydroxyethyl)piperazin-1-yl]methyl]-2-pyridyl]amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol -   (1R)-1-[2-[[5-[[4-(2-hydroxyethyl)piperazin-1-yl]methyl]-2-pyridyl]amino]-8-(propylamino)pyrido[3,4-d]pyrimidin-6-yl]ethanol -   N8-isopropyl-6-(3-methyloxetan-3-yl)-N2-(6-piperazin-1-ylpyridazin-3-yl)pyrido[3,4-d]pyrimidine-2,8-diamine -   N8-isopropyl-6-(3-methyloxetan-3-yl)-N2-[5-(piperazin-1-ylmethyl)-2-pyridyl]pyrido[3,4-d]pyrimidine-2,8-diamine -   6-(3-methyloxetan-3-yl)-N2-[5-(piperazin-1-ylmethyl)-2-pyridyl]-N8-[(3S)-tetrahydropyran-3-yl]pyrido[3,4-d]pyrimidine-2,8-diamine -   4-[6-[[6-[(1R)-1-hydroxyethyl]-8-[isopropyl(methyl)amino]pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]-1,4-diazepan-5-one -   (1R)-1-[8-(isopropylamino)-2-[(6-methyl-5-piperazin-1-yl-2-pyridyl)amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol -   (1R)-1-[2-[[6-(2-hydroxyethyl)-7,8-dihydro-5H-1,6-naphthyridin-2-yl]amino]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-6-yl]ethanol -   (1R)-1-[8-(isopropylamino)-2-[[6-[2-(methylamino)ethyl]-7,8-dihydro-5H-1,6-naphthyridin-2-yl]amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol -   N2-(6-piperazin-1-ylpyridazin-3-yl)-6-[(3S)-tetrahydrofuran-3-yl]-N8-[(3S)-tetrahydropyran-3-yl]pyrido[3,4-d]pyrimidine-2,8-diamine -   N2-[5-(piperazin-1-ylmethyl)-2-pyridyl]-6-[(3R)-tetrahydrofuran-3-yl]-N8-[(3S)-tetrahydropyran-3-yl]pyrido[3,4-d]pyrimidine-2,8-diamine -   (1R)-1-[2-[[6-[2-(dimethylamino)ethyl]-7,8-dihydro-5H-1,6-naphthyridin-2-yl]amino]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-6-yl]ethanol -   (2S)-1-[4-[[6-[[8-(ethylamino)-6-[(1R)-1-hydroxyethyl]pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]methyl]piperazin-1-yl]propan-2-ol -   (2R)-1-[4-[[6-[[8-(ethylamino)-6-[(1R)-1-hydroxyethyl]pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]methyl]piperazin-1-yl]propan-2-ol -   (1R)-1-[8-(isopropylamino)-2-[[5-[(2R)-2-methylpiperazin-1-yl]-2-pyridyl]amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol -   (1R)-1-[8-(isopropylamino)-2-[[5-[(2S)-2-methylpiperazin-1-yl]-2-pyridyl]amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol -   N8-isopropyl-N2-(5-piperazin-1-yl-2-pyridyl)-6-[(2S)-tetrahydrofuran-2-yl]pyrido[3,4-d]pyrimidine-2,8-diamine -   (1R)-1-[8-(cyclobutylamino)-2-[[5-[[4-(2-hydroxyethyl)piperazin-1-yl]methyl]-2-pyridyl]amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol -   (1R)-1-[8-(cyclopropylmethylamino)-2-[[5-[[4-(2-hydroxyethyl)piperazin-1-yl]methyl]-2-pyridyl]amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol -   6-(3-methyloxetan-3-yl)-N2-(5-piperazin-1-yl-2-pyridyl)-N8-propyl-pyrido[3,4-d]pyrimidine-2,8-diamine -   6-(3-methyloxetan-3-yl)-N2-[5-(piperazin-1-ylmethyl)-2-pyridyl]-N8-propyl-pyrido[3,4-d]pyrimidine-2,8-diamine -   N2-(5-piperazin-1-yl-2-pyridyl)-N8-propyl-6-tetrahydrofuran-3-yl-pyrido[3,4-d]pyrimidine-2,8-diamine -   N2-[5-(piperazin-1-ylmethyl)-2-pyridyl]-N8-propyl-6-tetrahydrofuran-3-yl-pyrido[3,4-d]pyrimidine-2,8-diamine -   N8-isopropyl-6-(3-methyloxetan-3-yl)-N2-(5-piperazin-1-yl-2-pyridyl)pyrido[3,4-d]pyrimidine-2,8-diamine -   N8-isopropyl-N2-(5-piperazin-1-yl-2-pyridyl)-6-tetrahydrofuran-3-yl-pyrido[3,4-d]pyrimidine-2,8-diamine -   2-[4-[[6-[[8-(isopropylamino)-6-tetrahydrofuran-3-yl-pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]methyl]piperazin-1-yl]ethanol -   2-[4-[[6-[[6-tetrahydrofuran-3-yl-8-[[(3S)-tetrahydropyran-3-yl]amino]pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]methyl]piperazin-1-yl]ethanol -   (1R)-1-[2-[[5-[[4-(hydroxymethyl)-1-piperidyl]methyl]-2-pyridyl]amino]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-6-yl]ethanol -   1-[[6-[[6-[(1R)-1-hydroxyethyl]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]methyl]piperidin-4-ol -   1-[[6-[[8-(tert-butylamino)-6-[(1R)-1-hydroxyethyl]pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]methyl]piperidin-4-ol -   (1R)-1-[8-(tert-butylamino)-2-[[5-[[4-(hydroxymethyl)-1-piperidyl]methyl]-2-pyridyl]amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol -   1-[[6-[[6-[(1R)-1-hydroxyethyl]-8-(isobutylamino)pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]methyl]piperidin-4-ol -   (1R)-1-[2-[[5-[[4-(hydroxymethyl)-1-piperidyl]methyl]-2-pyridyl]amino]-8-(isobutylamino)pyrido[3,4-d]pyrimidin-6-yl]ethanol -   1-[6-[[6-[(1R)-1-hydroxypropyl]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]piperazin-2-one -   (1R)-1-[2-[[5-[[4-(2-hydroxyethyl)piperazin-1-yl]methyl]-6-methyl-2-pyridyl]amino]-8-(propylamino)pyrido[3,4-d]pyrimidin-6-yl]ethanol     Aspect (25): A pharmaceutical composition comprising the compound or     pharmaceutically acceptable salt thereof according to any one of     Aspects (1) to (24) and a pharmaceutically acceptable carrier.     Aspect (26): A pharmaceutical composition exhibiting a CDK4/6     inhibitory activity, comprising the compound or pharmaceutically     acceptable salt thereof according to any one of Aspects (1) to (24)     as an active ingredient.     Aspect (27): A drug for prevention or treatment of rheumatoid     arthritis, arteriosclerosis, pulmonary fibrosis, cerebral     infarction, or cancer, the drug comprising the compound or     pharmaceutically acceptable salt thereof according to any one of     Aspects (1) to (24) as an active ingredient.

Advantageous Effects of Invention

The compound of the present invention exhibits a superior CDK4/6 inhibitory activity and is useful as a drug for prevention or treatment of rheumatoid arthritis, arteriosclerosis, pulmonary fibrosis, cerebral infarction, or cancer.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is graphs showing the results (scores) obtained through administration of the compound of the present invention to mice.

DESCRIPTION OF EMBODIMENTS

Now will be described the structures (groups) of the compound of the present invention represented by Formula (I). The description of “groups” with parentheses is as follows: For example, the term “(cycloalkyl)-alkyl” refers to a cycloalkyl group bonded to an alkyl group such that the alkyl group is bonded to a structure other than the cycloalkyl group. Similarly, the term “(heterocyclyl)-alkyl” refers to a heterocyclyl group bonded to an alkyl group such that the alkyl group is bonded to a structure other than the heterocyclyl group.

It must be noted that, as used herein and the annexed claims, the singular form “a”, “an” or “the” may include plural referents unless the context clearly dictates otherwise.

As used herein, “C₃₋₆ cycloalkyl group substituted with zero to two —OH groups, zero to two C₁₋₈ alkoxy groups, and zero to six fluorine atoms” refers to the case where the C₃₋₆ cycloalkyl group is substituted with the following substituents: zero to two —OH groups, zero to two C₁₋₈ alkoxy groups, and zero to six fluorine atoms. Examples of the substituted C₃₋₆ cycloalkyl group include a C₃₋₆ cycloalkyl group substituted with two —OH groups, one C₁₋₈ alkoxy group, and three fluorine atoms; a C₃₋₆ cycloalkyl group substituted with two C₁₋₈ alkoxy groups and four fluorine atoms; and a C₃₋₆ cycloalkyl group substituted with one —OH group, and the like. The C₃₋₆ cycloalkyl group is not substituted in the case where the number of all the substituents is zero.

As used herein, “C₁₋₈” refers to a group having one to eight carbon atoms, and “C₁₋₆” refers to a group having one to six carbon atoms. Similarly, “5- to 10-membered” refers to a structure having 5 to 10 carbon atoms, and “5- or 6-membered” refers to a structure having five or six carbon atoms.

Non-limiting examples of the groups described in this specification are as follows:

The term “alkyl” as used herein refers to a monovalent group obtained by removal of one hydrogen atom from an alkane at any carbon atom.

The term “alkylene” as used herein refers to a divalent group obtained by removal of two hydrogen atoms from an alkane at any two different carbon atoms.

The term “alkane” as used herein refers to a saturated aliphatic hydrocarbon.

The term “C₁₋₈ alkyl” as used herein refers to a linear or branched hydrocarbon group having one to eight carbon atoms. Examples of the C₁₋₈ alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, neopentyl, isopentyl, 1,2-dimethylpropyl, n-hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, isoheptyl, n-octyl, isooctyl, and the like.

The alkane of “C₁₋₈ alkylene” as used herein refers to a linear or branched hydrocarbon having one to eight carbon atoms. Examples of the alkane include methane, ethane, propane, n-butane, 2-methylpropane, n-pentane, 2,2-dimethylpropane, n-hexane, 2-methylpentane, 3-methylpentane, 2,2-dimethylbutane, 2,3-dimethylbutane, n-heptane, 2,2-dimethylhexane, 2,3-dimethylhexane, n-octane, 2-methylheptane, and the like.

The term “cycloalkyl” as used herein refers to a monovalent group obtained by removal of one hydrogen atom from a cycloalkane at any carbon atom.

The term “cycloalkylene” as used herein refers to a divalent group obtained by removal of two hydrogen atoms from a cycloalkane at any two different carbon atoms.

The term “cycloalkylidene” refers to a divalent group obtained by removal of two hydrogen atoms from a cycloalkane at any one carbon atom.

The term “cycloalkane” as used herein refers to an alicyclic hydrocarbon.

The cycloalkane of “C₃₋₁₂ cycloalkyl,” “C₃₋₁₂ cycloalkylene,” or “C₃₋₁₂ cycloalkylidene” as used herein refers to a monocyclic or polycyclic 3- to 12-membered aliphatic hydrocarbon ring. Specific examples of the cycloalkane include cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane, spiro[3.3]heptane, bicyclo[1.1.1]pentane, bicyclo[2.2.2]octane, adamantane, and the like.

The term “heterocyclyl” as used herein refers to a monovalent group obtained by removal of one hydrogen atom from a heterocycle at any carbon or nitrogen atom.

The term “heterocyclylene” as used herein refers to a divalent group obtained by removal of two hydrogen atoms from a heterocycle at any two different carbon or nitrogen atoms.

The term “heterocyclylidene” as used herein refers to a divalent group obtained by removal of two hydrogen atoms from a heterocycle at any one carbon atom.

The term “heterocycle” as used herein refers to a ring containing a heteroatom selected from sulfur, nitrogen, and oxygen atoms.

The heterocycle of “4- to 12-membered heterocyclyl,” “4- to 12-membered heterocyclylene,” or “4- to 12-membered heterocyclylidene” as used herein refers to “4- to 12-membered heterocycloalkane,” “4- to 12-membered heterocycloalkane” having an unsaturated bond, a 4- to 12-membered ring composed of a heterocycloalkane and a heteroarene or arene bonded to a portion of the heterocycloalkane, a 4- to 12-membered ring composed of a cycloalkane and a heteroarene bonded to a portion of the cycloalkane, a 4- to 12-membered ring containing a heteroatom and having a spiro structure, or a 4- to 12-membered ring containing a heteroatom and having a cross-linked structure. The term “4- to 12-membered heterocycloalkane” refers to a 4- to 12-membered cyclic heteroalkane; i.e., a monocyclic or polycyclic aliphatic hydrocarbon ring containing one to four heteroatoms selected from sulfur, nitrogen, and oxygen atoms. Specific examples of the “4- to 12-membered heterocycloalkane” include aziridine, thiirane, azetidine, oxetane, thietane, tetrahydrofuran, tetrahydropyran, 1,4-dioxane, piperidine, piperazine, pyrrolidine, imidazolidine, pyrazolidine, morpholine, thiomorpholine, tetrahydrothiopyran, tetrahydrothiophene, 1,4-diazepane, oxepane, and the like. A compound having a “spiro structure” is composed of two cyclic structures (cycloalkanes or heterocycloalkanes) that are bonded to one common carbon atom. Examples of the compound include 2-azaspiro[3.3]heptane, 1,6-diazaspiro[3.3]heptane, 2,6-diazaspiro[3.3]heptane, 2,6-diazaspiro[3.4]octane, 2,7-diazaspiro[3.5]nonane, 1,7-diazaspiro[4.5]decane, 2,8-diazaspiro[4.5]decane, 4,7-diazaspiro[2.5]octane, and the like. A compound having a “cross-linked structure” is composed of two cyclic structures (cycloalkanes and heterocycloalkanes) that are bonded to two or more common carbon, nitrogen, or oxygen atoms. Examples of the compound include 2,5-diazabicyclo[2.2.2]octane, 3,8-diazabicyclo[3.2.1]octane, 1,4-diazabicyclo[3.2.2]nonane, octahydropyrrolo[3,4-b]pyrrole, and the like.

The term “aryl” as used herein refers to a monovalent group obtained by removal of one hydrogen atom from an arene at any carbon atom.

The term “arylene” as used herein refers to a divalent group obtained by removal of two hydrogen atoms from an arene at any two different carbon atoms.

The term “arene” as used herein refers to an aromatic hydrocarbon.

The arene of “C₆₋₁₀ aryl” or “C₆₋₁₀ arylene” as used herein refers to an aromatic hydrocarbon ring having six to ten carbon atoms. Specific examples of the arene include benzene, naphthalene, and the like.

The term “heteroaryl” as used herein refers to a monovalent group obtained by removal of one hydrogen atom from a heteroarene at any carbon or nitrogen atom.

The term “heteroarylene” as used herein refers to a divalent group obtained by removal of two hydrogen atoms from a heteroarene at any two different carbon or nitrogen atoms.

The term “heteroarene” as used herein refers to an aromatic heterocyclic ring containing a heteroatom selected from sulfur, nitrogen, and oxygen atoms.

The heteroarene of “5- to 10-membered heteroaryl” or “5- to 10-membered heteroarylene” as used herein refers to a 5- to 10-membered aromatic heterocyclic ring containing one to four heteroatoms selected from sulfur, nitrogen, and oxygen atoms. Specific examples of the heteroarene include furan, thiophene, pyrrole, imidazole, pyrazole, triazole, tetrazole, thiazole, oxazole, isoxazole, oxadiazole, thiadiazole, isothiazole, pyridine, pyridazine, pyrazine, pyrimidine, quinolone, isoquinolone, benzofuran, benzothiophene, indole, indazole, benzimidazole, and the like.

The term “(4- to 12-membered heterocyclyl)-C₁₋₆ alkyl” as used herein refers to a 4- to 12-membered heterocyclyl group bonded to a C₁₋₆ alkyl group such that the C₁₋₆ alkyl group is bonded to a structure other than the 4- to 12-membered heterocyclyl group. Specific examples of the (4- to 12-membered heterocyclyl)-C₁₋₆ alkyl include groups prepared by bonding of any of the above-exemplified 4- to 12-membered heterocyclyl groups to any of the above-exemplified C₁₋₆ alkyl groups.

The term “(C₆₋₁₀ aryl)-C₁₋₆ alkyl” as used herein refers to a C₆₋₁₀ aryl group bonded to a C₁₋₆ alkyl group such that the C₁₋₆ alkyl group is bonded to a structure other than the C₆₋₁₀ aryl group. Specific examples of the (C₆₋₁₀ aryl)-C₁₋₆ alkyl include groups prepared by bonding of any of the above-exemplified C₆₋₁₀ aryl groups to any of the above-exemplified C₁₋₆ alkyl groups.

The term “(5- to 10-membered heteroaryl)-C₁₋₆ alkyl” as used herein refers to a 5- to 10-membered heteroaryl group bonded to a C₁₋₆ alkyl group such that the C₁₋₆ alkyl group is bonded to a structure other than the 5- to 10-membered heteroaryl group. Specific examples of the (5- to 10-membered heteroaryl)-C₁₋₆ alkyl include groups prepared by bonding of any of the above-exemplified 5- to 10-membered heteroaryl groups to any of the above-exemplified C₁₋₆ alkyl groups.

The term “C₁₋₈ alkylsulfonyl” as used herein refers to a C₁₋₈ alkyl group bonded to a sulfonyl (—S(═O)₂—) group such that the sulfonyl group is bonded to a structure other than the C₁₋₈ alkyl group.

The term “C₁₋₈ acyl” as used herein refers to a C₁₋₇ alkyl group bonded to a carbonyl (—CO—) group such that the carbonyl group is bonded to a structure other than the C₁₋₇ alkyl group.

The term “halogen” as used herein refers to a fluorine, chlorine, bromine, or iodine atom.

The term “C₁₋₈ alkoxy” as used herein refers to a linear, branched, or cyclic alkoxy group having one to eight carbon atoms. Specific examples of the C₁₋₈ alkoxy include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentyloxy, neopentyloxy, tert-pentyloxy, 2-methylbutoxy, n-hexyloxy, isohexyloxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, cycloheptyloxy, cyclooctyloxy, spiro[3.3]heptyloxy, bicyclo[2.2.2]octyloxy, and the like.

The term “alkenyl” as used herein refers to a monovalent group obtained by removal of one hydrogen atom from an alkene at any carbon atom.

The term “alkenylene” as used herein refers to a divalent group obtained by removal of two hydrogen atoms from an alkene at any two different carbon atoms.

The term “alkene” as used herein refers to an unsaturated aliphatic hydrocarbon having one double bond.

The term “C₂₋₈ alkenyl” as used herein refers to a chain aliphatic hydrocarbon having one double bond. Examples of the C₂₋₈ alkenyl include ethenyl (or vinyl), propenyl (or allyl), butenyl, and the like.

The term “alkynyl” as used herein refers to a monovalent group obtained by one hydrogen atom from an alkyne at any carbon atom.

The term “alkynylene” as used herein refers to a divalent group obtained by removal of two hydrogen atoms from an alkyne at any two different carbon atoms.

The term “alkyne” as used herein refers to an unsaturated aliphatic hydrocarbon having one triple bond.

The term “C₂₋₄ alkynyl” as used herein refers to a chain hydrocarbon group having one triple bond. Examples of the C₂₋₄ alkynyl include ethynyl, propynyl, butynyl, and the like.

L is preferably —NR⁵—.

The “C₁₋₆ alkyl” of R⁵ is preferably methyl or ethyl.

R⁵ is preferably a hydrogen atom or a methyl group.

The “C₁₋₈ alkyl” of R¹ is preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, neopentyl, isopentyl, 1,2-dimethylpropyl, n-hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, isoheptyl, n-octyl, or isooctyl.

The “C₃₋₁₂ cycloalkyl” of R¹ is preferably cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, spiro[3.3]heptyl, bicyclo[1.1.1]pentane, bicyclo[2.2.2]octyl, or adamantyl.

The “(C₃₋₁₂ cycloalkyl)-C₁₋₆ alkyl” of R¹ is preferably cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, or cyclopentylethyl.

The heterocycle of “4- to 12-membered heterocyclyl” in R¹ is preferably azetidine, oxetane, thietane, tetrahydrofuran, 1,4-dioxane, morpholine, thiomorpholine, tetrahydropyran, tetrahydrothiophene, or oxepane.

The “(4- to 12-membered heterocyclyl)-C₁₋₆ alkyl” of R¹ is preferably (tetrahydrofuranyl)methyl, (tetrahydropyranyl)methyl, (tetrahydrofuranyl)ethyl, or (tetrahydropyranyl)ethyl.

The “C₆₋₁₀ aryl” of R¹ is preferably phenyl.

The “(C₆₋₁₀ aryl)-C₁₋₆ alkyl” of R¹ is preferably phenylmethyl or phenylethyl.

The “5- to 10-membered heteroaryl” of R¹ is preferably furanyl, pyrazolyl, or thienyl.

The “halogen” in the substituent of R¹ is preferably a fluorine or chlorine atom.

The “—COOR⁶” in the substituent of R¹ is preferably —COOH or —COOCH₃.

The “R⁷” in the substituent of R¹ is preferably ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, neopentyl, isopentyl, 1,1-dimethyl-2-methoxyethyl, 1-methyl-2-methoxyethyl, 1-methyl-2-hydroxyethyl, 2,2,2-trifluoroethyl, hydroxymethyl, or 1-methyl-2,2,2-trifluoroethyl.

The “C₃₋₆ cycloalkyl optionally substituted with a substituent selected from the group consisting of one or two —OH groups, one or two C₁₋₈ alkoxy groups, and one to six fluorine atoms” in the substituent of R¹ is preferably cyclopentyl, cyclohexyl, 4-methoxycyclohexyl, or 4-isopropoxycyclohexyl.

The 3- to 10-membered heterocyclyl optionally substituted with a substituent selected from the group consisting of one or two —OH groups, one or two C₁₋₈ alkoxy groups, and one to six fluorine atoms in the substituent of R¹ is preferably tetrahydrofuranyl, tetrahydropyranyl, or 2,2-dimethyltetrahydropyranyl.

R¹ preferably has any of the following structures:

The “C₁₋₈ alkyl” of R² is preferably methyl, ethyl, or n-propyl, and the substituent is preferably a hydroxy, methoxy, or ethoxy group or a fluorine atom. The “4- to 6-membered heterocyclyl” of R² is preferably oxetane or tetrahydrofuranyl.

The “C₁₋₈ acyl” of R² is preferably acetyl.

The “—COOR⁸” of R² is preferably —COOH or —COOCH₃.

The “—CONR⁹R¹⁰” of R² is preferably —CON(CH₃)₂.

R⁹ and R¹⁰ of —CONR⁹R¹⁰ of R² may be bonded via a single bond or —O— to form a ring including the nitrogen atom bonded to R⁹ and R¹⁰. Examples of such a ring include the following structures:

R² preferably has any of the following structures:

The “C₁₋₈ alkyl” of R³ is preferably methyl.

The “halogen” of R³ is preferably a fluorine or chlorine atom.

R³ is preferably a hydrogen, fluorine, or chlorine atom or a methyl group.

X, Y, and Z preferably correspond to any of the following combinations: X, Y, and Z are each CH; X is a nitrogen atom and Y and Z are each CH; Y is a nitrogen atom and X and Z are each CH; and Z is a nitrogen atom and X and Y are each CH.

The “C₁₋₈ alkylene” of A¹ is preferably methylene, ethylene, or n-propylene.

The structure obtained by replacement of one or two sp³ carbon atoms at any positions of A¹ is preferably —O—, —OCH₂—, —OCH₂CH₂—, —OCH₂CH₂CH₂—, —CH₂O—, —CH₂OCH₂—, —CH₂OCH₂CH₂—, —CH₂CO—, —COCH₂—, —CH₂CH₂CO—, —COCH₂CH₂—, —CH₂COCH₂—, —CH₂COCH₂CH₂—, —NR¹⁴—, —NR⁴CH₂—, —CH₂NR¹⁴—, —NR¹⁴CH₂CH₂—, —CH₂NR¹⁴CH₂—, or —CH₂CH₂NR¹⁴—.

The “C₁₋₇ alkylene” of A² is preferably methylene, ethylene, or n-propylene.

The “C₃₋₁₂ cycloalkylene” of A² is preferably cyclopropylene, cyclobutylene, cyclopentylene, or cyclohexylene.

The heterocycle of “4- to 12-membered heterocyclylene” of A² is preferably piperidine, piperazine, pyrrolidine, morpholine, tetrahydrofuran, tetrahydropyran, 1,4-diazepane, oxepane, 2-azaspiro[3.3]heptane, 1,6-diazaspiro[3.3]heptane, 2,6-diazaspiro[3.3]heptane, 2,6-diazaspiro[3.4]octane, 2,5-diazabicyclo[2.2.2]octane, 3,8-diazabicyclo[3.2.1]octane, 2,7-diazaspiro[3.5]nonane, 1,7-diazaspiro[4.5]decane, 2,8-diazaspiro[4.5]decane, 4,7-diazaspiro[2.5]octane, 1,4-diazabicyclo[3.2.2]nonane, or octahydropyrrolo[3,4-b]pyrrole.

The heterocycle of “4- to 12-membered heterocyclylidene” of A² is preferably oxetane, tetrahydrofuran, tetrahydropyran, pyrrolidine, piperidine, piperazine, morpholine, or oxepane.

The “C₆₋₁₀ arylene” of A² is preferably phenylene.

The heteroarene of “5- to 10-membered heteroarylene” of A² is preferably furan, thiophene, pyrrole, imidazole, pyrazole, triazole, tetrazole, thiazole, oxazole, isoxazole, oxadiazole, thiadiazole, isothiazole, pyridine, pyridazine, pyrazine, pyrimidine, quinolone, isoquinoline, benzofuran, benzothiophene, indole, indazole, or benzimidazole.

The “halogen” of A³ is preferably a fluorine or chlorine atom.

The “—R²⁵” of A³ is a hydrogen atom or a methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl group. The —R²⁵ substituted with a substituent is preferably a hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 2-hydroxy-2-propyl, 2-hydroxy-1-propyl, 1-hydroxy-2-propyl, 1-hydroxy-2-methyl-2-propyl, 2-hydroxy-2-methyl-1-propyl, trifluoromethyl, 2,2,2-trifluoroethyl, carboxymethyl, 1-carboxyethyl, 2-carboxyethyl, 2-carboxy-2-propyl, or cyanomethyl group.

The “—OR²⁶” of A³ is preferably —OH, methoxy, ethoxy, or isopropoxy.

The “—NR²⁷R²⁸” of A³ is preferably amino, dimethylamino, methylamino, pyrrolidin-1-yl, piperidin-1-yl, piperazin-1-yl, or morpholin-1-yl.

The “—C(═O)R²⁹” of A³ is preferably acetyl. The —C(═O)R²⁹ substituted with a substituent is preferably hydroxyacetyl.

The “—C(═O)—OR³⁰” of A³ is preferably —COOH, methoxycarbonyl, ethoxycarbonyl, or isopropoxycarbonyl.

The “—C(═O)—NR³⁴R³⁵” of A³ is preferably aminocarbonyl (or carbamoyl), (methylamino)carbonyl, (dimethylamino)carbonyl, (pyrrolidin-1-yl)carbonyl, (piperidin-1-yl)carbonyl, (morpholin-1-yl)carbonyl, or (piperazin-1-yl)carbonyl.

The “—S(═O)₂—R⁴⁰” of A³ is preferably methanesulfonyl or ethylsulfonyl.

R¹⁴ to R⁴⁴ in A¹, A², and A³ may be bonded in A¹, A², or A³ or between A¹ and A², between A¹ and A³, or between A² and A³ via a single bond, —O—, —NR⁵⁰—, or —S(═O)_(p)— to form a ring. Examples of such a ring include the following structures:

R¹¹ or R¹³ may be bonded to A¹, A², or A³ via a single bond, —O—, —NR⁵¹—, or —S(═O)_(p)— to form a ring. Examples of such a ring include the following structures:

Preferred examples of the aforementioned entire structure are as follows:

A preferred compound represented by Formula (I) is composed of a combination of a group selected from the above-defined ones and a preferred group, or a combination of preferred groups.

The compound of the present invention represented by Formula (I) may optionally be formed into a pharmaceutically acceptable salt. Examples of the salt include salts with inorganic acids, such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, carbonic acid, and the like; salts with organic acids, such as formic acid, acetic acid, propionic acid, trifluoroacetic acid, phthalic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like; salts with amino acids, such as lysine, arginine, ornithine, glutamic acid, aspartic acid, and the like; salts with alkali metals, such as sodium, potassium, lithium, and the like; salts with alkaline earth metals, such as calcium magnesium, and the like; salts with metals, such as aluminum, zinc, iron, and the like; salts with organic bases, such as methylamine, ethylamine, t-octylamine, diethylamine, trimethylamine, triethylamine, ethylenediamine, piperidine, piperazine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N-methylglucamine, tris(hydroxymethyl)aminomethane, N,N′-dibenzylethylenediamine, and the like; and ammonium salts and the like.

The present invention also encompasses compounds prepared through replacement of one or more atoms of the compound represented by Formula (I) with stable isotopes or radioisotopes.

The present invention also encompasses stereoisomers, racemates, and all acceptable optical isomers of the compound represented by Formula (I).

Tautomers of the compound of the present invention may be generated depending on the combination of substituents. The present invention also encompasses such tautomers.

Now will be described a typical process for synthesizing the compound of the present invention represented by Formula (I).

The compound of the present invention can be synthesized by the process described below. R¹, R³, R⁴, and R⁷ shown in the following reaction schemes are as defined in Formula (I). The reagents or solvents and the like shown in the reaction schemes are for illustrative purposes only as described below. Each substituent may optionally be protected with an appropriate protective group or deprotected in an appropriate step (reference: PROTECTIVE GROUPS in ORGANIC SYNTHESIS, 4TH EDITION, John Wiley & Sons, Inc.). The abbreviations of substituents, reagents, and solvents described below and in tables are as follows:

Me: methyl

Et: ethyl

Ph: phenyl

Boc: tert-butoxycarbonyl

Cbz: benzyloxycarbonyl

THF: tetrahydrofuran

DMF: N,N-dimethylformamide

NMP: N-methylpyrrolidone

TFA: trifluoroacetic acid

TBS: tert-butyldimethylsilyl

BINAP: 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl

TBDPS: tert-butyldiphenylsilyl

DIPEA: N,N-Diisopropylethylamine

LAH: Lithium aluminium hydride

DMAP: 4-Dimethylaminopyridine

Ac: acetyl

Ms: mesyl

WSC: water-soluble carbodiimide (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide)

m-CPBA: m-chloroperoxybenzoic acid

DAST: diethylaminosulfur trifluoride

dba: dibenzylideneacetone

DIBAL-H: diisobutylaluminium hydride

1) Synthesis of Compound I-e

Compound I-e, which is a known compound, can be synthesized by any process known to those skilled in the art; for example, the aforementioned process.

2) Synthesis of Compound I-f from Compound I-e

Compound I-e is reacted with a terminal alkyne derivative represented by the formula R²—C≡CH in an appropriate organic solvent (e.g., THF or DMF) in the presence of an appropriate palladium catalyst (e.g., tetrakis(triphenylphosphin)palladium), appropriate copper catalyst (e.g., copper iodide (I)) and appropriate base (e.g., triethylamine) at a temperature of 0° C. to the reflux temperature of the solvent, to yield compound I-f.

3) Synthesis of Compound I-h from Compound I-f

Compound I-f is reacted with hydroxylamine or a salt thereof in an appropriate organic solvent (e.g., ethanol) in the presence or absence of an appropriate base (e.g., sodium acetate) at a temperature of 0° C. to the reflux temperature of the solvent. The resultant hydroxyimine compound is reacted with an appropriate acid or base (e.g., silver triflate or potassium carbonate) to yield compound I-h.

4) Synthesis of Compound I-i from Compound I-h

Compound I-h is reacted with an appropriate halogenating agent (e.g., thionyl chloride) in an appropriate organic solvent (e.g., dichloromethane) or under solvent-free conditions at a temperature of 0° C. to 140° C., to yield compound I-i.

5) Synthesis of Compound I-j from Compound I-i

Compound I-i is reacted with an amine, alcohol, or thiol derivative represented by the formula R¹-L-H in an appropriate organic solvent (e.g., THF or 1,4-dioxane) or under solvent-free conditions in the presence or absence of an appropriate base (e.g., triethylamine, potassium carbonate, or sodium hydride) at a temperature of 0° C. to the reflux temperature of the solvent, to yield compound I-j.

In this step, R² may be modified by any process known to those skilled in the art in view of the intended structure of the compound.

6) Synthesis of Compound I-k from Compound I-j

Compound I-j is reacted with an appropriate oxidant (e.g., Oxone® or m-chloroperbenzoic acid) in an appropriate organic solvent (e.g., dichloromethane or water) at a temperature of 0° C. to the reflux temperature of the solvent, to yield compound I-k.

7) Synthesis of Compound I-l from Compound I-k

Compound I-k is reacted with an appropriate halogenating agent (e.g., N-chlorosuccinimide) in an appropriate organic solvent (e.g., dichloromethane or 1,2-dichloroethane) at a temperature of 0° C. to the reflux temperature of the solvent, to yield compound I-l.

In this step, R³ may be modified by any process known to those skilled in the art in view of the intended structure of the compound.

8) Synthesis of Compound I-m from Compound I-l

Compound I-l is reacted with an amine derivative represented by the formula R⁴-(nitrogen-containing heteroaryl with X, Y, and Z) —NH₂ of the above scheme in an appropriate organic solvent (e.g., NMP, THF, or toluene) or under solvent-free conditions in the presence or absence of an appropriate base (e.g., sodium hydride, triethylamine, or N,N-diisopropyl-N-ethylamine) at a temperature of 0° C. to the reflux temperature of the solvent, to yield compound I-m.

If L, R¹, R², or R⁴ of compound I-m is protected with an appropriate protective group, deprotection can be performed by any process known to those skilled in the art. For example, deprotection can be performed through reaction of the compound with an appropriate deprotecting reagent (e.g., TFA or hydrogen chloride for a Boc protective group, lithium hydroxide for a benzoyl protective group, or hydrogen in the presence of Pd/C for a Cbz protective group) in an appropriate organic solvent (e.g., dichloromethane, methanol, or THF) or under solvent-free conditions at a temperature of 0° C. to the reflux temperature of the solvent (reference: Green's Protective Groups in Organic Synthesis, 4th edition, John Wiley & Sons Inc.).

If compound I-m is protected with two or more protective groups, deprotection may be performed in an appropriate order depending on the structure of compound I-m.

In each of the reactions 9) to 13) described below, L, R¹, R², or R⁴ of compound I-m is appropriately protected depending on the corresponding reaction conditions. After completion of the reaction, deprotection can be performed by an appropriate process.

9) Synthesis of Compound I-n from Compound I-m

Compound I-m in which R⁴ has a primary or secondary amine structure is reacted with an optionally substituted epoxide in an appropriate organic solvent (e.g., dichloromethane, NMP, or THF) in the presence or absence of an appropriate acid (e.g., boron trifluoride-diethyl ether complex) or an appropriate base (e.g., potassium carbonate or triethylamine) at a temperature of 0° C. to the reflux temperature of the solvent, to yield compound I-n.

10) Synthesis of Compound I-o from Compound I-m

Compound I-m in which R⁴ has a primary or secondary amine structure is reacted with a carboxylic acid chloride, a carboxylic anhydride, or a carboxylic acid and a condensation reagent in an appropriate organic solvent (e.g., NMP, THF, or pyridine) in the presence or absence of an appropriate base (e.g., triethylamine or N,N-diisopropyl-N-ethylamine) at a temperature of 0° C. to the reflux temperature of the solvent, to yield compound I-o.

11) Synthesis of Compound I-p from Compound I-m

Compound I-m in which R⁴ has a primary or secondary amine structure is reacted with sulfonic acid chloride in an appropriate organic solvent (e.g., NMP, THF, or pyridine) in the presence or absence of an appropriate base (e.g., triethylamine or N,N-diisopropyl-N-ethylamine) at a temperature of 0° C. to the reflux temperature of the solvent, to yield compound I-p.

12) Synthesis of Compound I-q from Compound I-m

Compound I-m in which R⁴ has a primary or secondary amine structure is reacted with an optionally substituted ketone or aldehyde and an appropriate reductant (e.g., sodium triacetoxyborohydride or sodium cyanoborohydride) in an appropriate organic solvent (e.g., NMP or methanol) in the presence of an appropriate acid (e.g., acetic acid) at a temperature of room temperature to the reflux temperature of the solvent, to yield compound I-q.

13) Synthesis of Compound I-r from Compound I-m

Compound I-m in which R⁴ has a primary or secondary amine structure is reacted with a compound having a leaving group (e.g., a halogen atom or a sulfonyloxy group) in an appropriate organic solvent (e.g., NMP, THF, or pyridine) in the presence or absence of an appropriate base (e.g., triethylamine or N,N-diisopropyl-N-ethylamine) at a temperature of 0° C. to the reflux temperature of the solvent, to yield compound I-r.

14) Synthesis of Compound I-s from Compound I-m

Compound I-m in which R⁴ has a primary or secondary amine structure is reacted with a compound having a structure of Michael acceptor in an appropriate organic solvent (e.g., methanol, THF) at a temperature of 0° C. to the reflux temperature of the solvent to yield compound I-s.

The compound of the present invention exhibits a CDK4/6 inhibitory activity and thus is useful for the prevention or treatment of a disease associated with CDK4/6. Specifically, the compound is useful for the treatment of rheumatoid arthritis, arteriosclerosis, pulmonary fibrosis, cerebral infarction, or cancer and the protection of bone marrow. In particular, the compound is effective for the treatment of rheumatoid arthritis or cancer and the protection of bone marrow.

The compound of the present invention preferably exhibits selectivity for the CDK4/6 inhibitory activity compared to the inhibitory activity against another cyclin-dependent kinase, such as CDK2 inhibitory activity. Such selectivity of the compound is expected to reduce the expression of genotoxicity because the inhibition of CDK2 is also involved in DNA replication. Preferably, the compound of the present invention selectively inhibits CDK4 rather than CDK2.

The active ingredient of the present invention may be provided in any preparation form, such as a solid, semisolid, or liquid form, and the like. The active ingredient may be provided in any dosage form, such as an oral form or a parenteral form (e.g., an injection, a transdermal agent, an eye drop, a suppository, a nasal agent, or an inhalant, and the like).

A drug containing the active ingredient of the present invention is prepared with a common additive used for drug preparation. Examples of the additive for solid drugs include excipients, such as lactose, sucrose, glucose, cornstarch, potato starch, crystalline cellulose, light silicic anhydride, synthetic aluminum silicate, magnesium aluminometasilicate, calcium hydrogen phosphate, and the like; binders, such as crystalline cellulose, carboxymethyl cellulose, hydroxypropyl cellulose, sodium carboxymethyl cellulose, poly(vinylpyrrolidone), and the like; disintegrants, such as starch, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, croscarmellose sodium, sodium carboxymethyl starch, and the like; lubricants, such as talc stearic acid, and the like; coating agents, such as hydroxymethyl propyl cellulose, hydroxypropyl methyl cellulose phthalate, ethyl cellulose, and the like; and colorants. Examples of the additive for semisolid drugs include bases, such as white vaseline, and the like. Examples of the additive for liquid drugs include solvents, such as ethanol, and the like; solubilizers, such as ethanol, and the like; preservatives, such as paraoxybenzoic acid esters, and the like; isotonic agents, such as glucose, and the like; buffers, such as citric acid, and the like; antioxidants, such as L-ascorbic acid, and the like; chelators, such as EDTA, and the like; suspending agents and emulsifiers, such as polysorbate 80, and the like; and the like.

The dose of the active ingredient of the present invention is typically about 1 to 1,000 mg/day. The active ingredient is typically administered once to three times a day.

EXAMPLES

The present invention will now be described in detail by way of Examples, which should not be construed as limiting the invention.

The structure of an isolated novel compound was determined by ¹H-NMR and/or mass spectrometry with a single quadrupole instrumentation equipped with an electron spray source, and other appropriate analytical methods. Chemical shifts (δ: ppm) and coupling constants (J: Hz) are shown for the ¹H-NMR spectra (400 MHz, DMSO-d₆, CD₃OD or CDCl₃). Abbreviations are as follows: s (singlet), d (doublet), t (triplet), q (quartet), brs (broad singlet), and m (multiplet). For the results of mass spectrometry, measurements are represented by (M+H)⁺; i.e., a value corresponding to a proton (H⁺) attached to the molecular mass (M) of a compound.

Reference Example 1 Synthesis of 5-bromo-2-(methylthio)pyrimidine-4-carboxylic acid

Mucobromic acid (300 g, 1.16 mol) was added to an aqueous solution (2.5 L) of 2-methyl-2-pseudothiourea sulfate (324 g, 1.16 mol) at room temperature. The resultant suspension was cooled to 0° C. with stir, and triethylamine (486 mL, 3.49 mol) was added dropwise thereto over four hours. The resultant reaction mixture was stirred overnight, and the completion of the reaction was confirmed by silica gel TLC. The reaction mixture was then acidified with concentrated hydrochloric acid (about 250 mL). The resultant yellow solid was collected by filtration and washed twice with water (500 mL) and then twice with diethyl ether (500 mL). The solid was dried under reduced pressure to yield the title compound (160 g, 55%).

Reference Example 2 Synthesis of methyl 5-bromo-2-methylthiopyrimidine-4-carboxylate

A solution of 5-bromo-2-(methylthio)pyrimidine-4-carboxylic acid (110 g, 0.44 mol) in methanol (1.1 L) was cooled to 0° C. with stir, and thionyl chloride (50 mL, 0.66 mol) was added dropwise thereto. The resultant reaction mixture was slowly heated, and the reaction was allowed to proceed under reflux for four hours. The completion of the reaction was confirmed by LC/MS and TLC, and the reaction mixture was cooled to room temperature. The volatiles were removed through evaporation under reduced pressure, and the residue was dissolved in ethyl acetate (1 L). The resultant solution was washed three times with 10% aqueous sodium carbonate solution (200 mL) and then twice with saturated brine (200 mL). The resultant organic phase was dried over anhydrous magnesium sulfate, and solid was separated by filtration. The filtrate was then concentrated under reduced pressure, and the resultant crude product was purified by silica gel column chromatography to yield the title compound (88 g, 75%).

Reference Example 3 Synthesis of Mixture of 5-bromo-2-methylthiopyrimidine-4-carbaldehyde and (5-bromo-2-methylthiopyrimidin-4-yl)methoxymethanol

A solution (375 mL) of methyl 5-bromo-2-methylsulfanylpyrimidine-4-carboxylate (25 g, 95 mmol) in THF was cooled to −78° C. and stirred under a nitrogen atmosphere. DIBAL-H (84 mL, 143 mmol, 1.7M toluene solution) was added dropwise to the THF solution, and the mixture was stirred at −78° C. for four hours. The completion of the reaction was confirmed by TLC, and the reaction was quenched through dropwise addition of methanol at −78° C. The resultant reaction mixture was allowed to warm slowly to 0° C. and diluted with ethyl acetate, and the mixture was filtrated through celite. The filtrate was washed twice with saturated brine (200 mL), and the resultant organic phase was dried over anhydrous magnesium sulfate. The resultant solid was separated by filtration, and the filtrate was concentrated to yield the title compound mixture (25 g, crude product). The crude product was used for the subsequent reaction without further purification.

Reference Example 4 Synthesis of tert-butyl 4-(6-nitropyridin-3-yl)piperazine-1-carboxylate

A mixture of 5-Bromo-2-nitropyridine (203 g, 1.37 mol), piperazine (153 g, 1.77 mol), tetrabutylammonium iodide (25.2 g, 0.068 mol), and potassium carbonate (207 g, 1.50 mol) in dimethyl sulfoxide (2.6 L) was stirred at 80° C. overnight. The resultant reaction mixture was cooled to room temperature, and the mixture was poured into water (7 L). The resultant solid was collected by filtration, and the solid was washed with dichloromethane (1 L×2) and dried. The filtrate was extracted with chloroform (2 L×7). The resultant organic phase was washed with water (2 L) and then with saturated brine (2 L), and the organic phase was concentrated under reduced pressure to yield solid. The resultant solid products were combined together and used for the subsequent reaction without further purification.

The solid product (490 g) was dissolved in THF (2 L) and water (500 mL), and sodium hydrogen carbonate (119 g, 1.42 mol) was added to the solution. To the resultant suspension was added di-tert-butyl dicarboxylate (262 g, 1.2 mol), and the mixture was stirred at room temperature for three hours. The reaction mixture was concentrated under reduced pressure, and the residue was diluted with water (1 L) and extracted with dichloromethane (1 L×3). The resultant organic phases were combined together and then washed with water (1 L). The aqueous phase was extracted with dichloromethane (300 mL). The resultant organic phases were combined together and dried over anhydrous magnesium sulfate. The solid was separated by filtration, and the filtrate was concentrated under reduced pressure. The resultant solid was suspended in ethyl acetate (2 L) and heated to 60° C., and the solid was separated by filtration at 60° C. The solid was dried under reduced pressure to yield the title compound (191 g, 62%)

APCI-MS (M+H)⁺309.1, C₁₋₄H₂₀N₄O₄=308.15

¹H-NMR δ(400 MHz, CDCl₃): 8.16 (d, J=9 Hz, 1H), 8.11 (d, J=3 Hz, 1H), 7.19 (dd, J=9.3 Hz, 1H), 3.64-3.61 (m, 4H), 3.45-3.42 (m, 4H), 1.47 (s, 9H).

Reference Example 5 Synthesis of tert-butyl 4-(6-aminopyridin-3-yl)piperazine-1-carboxylate

The tert-butyl 4-(6-nitropyridin-3-yl)piperazine-1-carboxylate synthesized in Reference Example 4 (83 g, 269 mmol) was dissolved in methanol (1.3 L) in Parr Shaker and Raney nickel (15 g, 50% aqueous suspension) was added thereto. The resultant reaction mixture was stirred under a hydrogen atmosphere (50 psi) for five hours. The reaction mixture was filtered through a Celite pad to separate solid, and the filtrate was concentrated under reduced pressure. The resultant solid was suspended in diethyl ether (120 mL) and stirred for four hours. Heptane was added to the suspension and cooled at 0° C. for 45 minutes. The resultant solid was separated by filtration and dried under reduced pressure to yield the title compound (62.5 g, 83%).

ESI-MS (M+H)⁺ 279, C₁₄H₂₂N₄O₂=278.17

Intermediates A-1 to A-44 were each synthesized by the process of Reference Example 4 and/or 5 with the corresponding halopyridine derivatives and amine derivatives. Appropriate protection or deprotection was performed as needed.

Reference Example 6 Synthesis of 6-aminopyridine-3-carbaldehyde

6-Aminopyridine-3-carbonitrile (1.9 g, 16 mmol) was dissolved in THF (160 mL) and cooled to −78° C. with stir. Diisobutylaluminium hydride (106.5 mL, 1.5M toluene solution) was slowly added dropwise to the solution at −78° C. and the mixture was allowed to warm to 20° C. with stir, followed by further stirring for two hours. The reaction was quenched by addition of ice water (100 mL) to the resultant reaction mixture, and the mixture was extracted three times with dichloromethane (50 mL). The resultant organic phases were combined together and then washed once with brine (100 mL) and dried over anhydrous sodium sulfate. The solid was separated by filtration, and the filtrate was concentrated under reduced pressure. The residue was roughly purified by silica gel column chromatography to yield a crude product of the title compound (1.7 g). The crude product was used for the subsequent reaction without further purification.

Reference Example 7 Synthesis of tert-butyl 4-[(6-aminopyridin-3-yl)methyl]piperazine-1-carboxylate

The crude 6-aminopyridine-3-carbaldehyde synthesized in Reference Example 6 (1.7 g, 13.9 mmol) and tert-butyl piperazine-1-carboxylate (3.2 g, 17.2 mmol) were dissolved in dichloromethane (50 mL) and stirred at room temperature for eight hours. To the resultant mixture was added sodium triacetoxyborohydride (8.84 g, 40.9 mmol) and stirred at room temperature for two hours. The reaction was monitored by LC/MS. After completion of the reaction, the reaction was quenched through addition of saturated aqueous sodium carbonate solution (50 mL), and the reaction mixture was extracted three times with ethyl acetate (50 mL). The resultant organic phases were combined together, and the mixture was washed once with brine (100 mL) and dried over anhydrous sodium sulfate. The resultant solid was separated by filtration, and then the filtrate was concentrated under reduced pressure. The residue was roughly purified by silica gel column chromatography to yield the title compound (3.3 g, 81%).

Reference Example 8 Synthesis of di-tert-butyl (5-methylpyridin-2-yl)imidodicarbonate

In reference to the process disclosed in WO2010/141406, 5-methylpyridine-2-amine (20 g, 185 mmol) and di-tert-butyl dicarbonate (101 g, 462 mmol) were dissolved in THF (160 mL) and 4-N,N-dimethylaminopyridine (3.6 g, 29.7 mmol) was added to the solution. The resultant reaction mixture was stirred at room temperature for three days. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate and washed with water. The resultant organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The solid was separated by filtration, and the filtrate was concentrated. The resultant solid was dissolved in ethyl acetate (50 mL) and heptane (50 mL) was added thereto. The solid was collected by filtration and dried under reduced pressure, to yield the title compound (25.1 g, 44%). The filtrate was concentrated, and the residue was purified by silica gel column chromatography to yield the title compound (17.9 g, 31%).

Reference Example 9 Synthesis of di-tert-butyl [5-(bromomethyl)pyridin-2-yl]imidodicarbonate

The di-tert-butyl (5-methylpyridin-2-yl)imidodicarbonate synthesized in Reference Example 8 (17.2 g, 55.8 mmol), N-bromosuccinimide (12.17 g, 68.4 mmol), and benzoyl peroxide (1.5 g, 8.1 mmol) were dissolved in carbon tetrachloride (100 mL) and the reaction was stirred at 80° C. for six hours. The reaction mixture was cooled to room temperature, and the resultant solid was separated by filtration. The filtrate was then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to yield a mixture of the title compound, di-tert-butyl [5-(dibromomethyl)pyridin-2-yl]imidodicarbonate, and di-tert-butyl (5-methylpyridin-2-yl)imidodicarbonate (14.5 g, 60.3:4.4:35.3, determined by the 1H-NMR spectrum). The mixture was used for the subsequent reaction without further purification.

Reference Example 10

Di-tert-butyl [5-(bromomethyl)pyridin-2-yl]imidodicarbonate (1 equivalent) was dissolved in DMF and an appropriate amine derivative (1.5 equivalents) and N,N-diisopropyl-N-ethylamine (3 equivalents) was added to the solution at room temperature. The reaction mixture was stirred at room temperature for several hours, and the mixture was then diluted with ethyl acetate and washed with saturated brine. The resultant organic phase was dried over anhydrous sodium sulfate, and the solid was separated by filtration. The filtrate was then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to yield a target amine derivative.

Reference Example 11

To the compound synthesized in Reference Example 10 was added an excess amount of trifluoroacetic acid and stirred at room temperature for several hours. The reaction mixture was concentrated under reduced pressure, and the resultant TFA salt of the target product was dissolved in methanol and applied onto a strong cation exchange resin (SCX). The SCX column was washed with methanol and the target product was eluted with ammonia (2 mol/L, methanol solution). The eluate was concentrated under reduced pressure to yield a target 2-aminopyridine derivative. The resultant product was used for the subsequent reaction without further purification.

In the case of the presence of a primary or secondary amino group in the compound besides the aminopyridine structure, the crude product was dissolved in THF and reacted with di-tert-butyl dicarbonate at room temperature. After completion of the reaction, the solvent was removed through evaporation, and the residue was roughly purified by silica gel column chromatography to yield a 2-aminopyridine derivative having a primary or secondary amino group protected with a Boc group.

Intermediates B-1 to B-68 were each synthesized by any of the processes of Reference Example 6 and/or 7 or Reference Examples 8 to 11 or a combination of the processes with the corresponding aldehyde or alkyl halide derivatives and amine derivatives. Appropriate protection or deprotection was performed as needed.

Reference Example 12

An appropriate amide derivative (1 equivalent) was dissolved in DMF, and sodium hydride (1 equivalent) was gradually added thereto at 0° C. and the mixture was stirred at room temperature for several minutes. The resultant reaction mixture was cooled to 0° C. and di-tert-butyl [5-(bromomethyl)pyridin-2-yl]imidodicarbonate (1.5 equivalents) was gradually added to the mixture. The reaction mixture was stirred at room temperature for several hours and then water was added to the mixture to stop the reaction. The mixture was extracted with ethyl acetate and washed with saturated brine. The resultant organic phase was dried over anhydrous sodium sulfate, and the solid was separated by filtration. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to yield a target amide derivative.

The following intermediates C-1 to C-5 were synthesized by the process of Reference Example 12 and 11 with the corresponding alkyl halide derivatives, amide derivatives, or urea derivatives. Appropriate protection or deprotection was performed as needed.

Reference Example 13 Synthesis of tert-butyl 4-(6-nitropyridin-3-yl)-3-oxopiperazine-1-carboxylate

In reference to the process disclosed in WO2012/031004, 2-nitro-5-bromopyridine (1.01 g, 5.0 mmol), tert-butyl 2-oxo-4-piperazinecarboxylate (1.00 g, 5.0 mmol, and cesium carbonate (3.26 g, 10.0 mmol) were suspended in 1,4-dioxane, and the suspension was bubbled with nitrogen gas for 30 minutes. To the suspension was added Xantphos (246 mg, 0.43 mmol) and tris(dibenzylideneacetone)dipalladium (229 mg, 0.25 mmol), and the mixture was stirred under reflux for two hours. The resultant reaction mixture was cooled to room temperature, and water and ethyl acetate were then added to the mixture, followed by filtration with Celite. The organic phase was separated from the filtrate, and the aqueous phase was extracted with ethyl acetate. The resultant organic phases were combined together and dried over anhydrous sodium sulfate, and the resultant solid was separated by filtration. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to yield the title compound (1.08 g, 67%).

¹H-NMR (CDCl₃) δ: 8.67 (1H, d, J=2.4 Hz), 8.32 (1H, d, J=8.8 Hz), 8.15 (1H, dd, J=8.8, 2.4 Hz), 4.33 (2H, s), 3.93-3.83 (4H, m), 1.51 (9H, s).

Reference Example 14 Synthesis of tert-butyl 4-(6-aminopyridin-3-yl)-3-oxopiperazine-1-carboxylate

The compound synthesized in Reference Example 13 (1.08 g, 3.34 mmol) was dissolved in ethanol (45 mL) and THF (22 mL). Palladium-carbon (108 mg) was added to the solution, and the mixture was stirred under a hydrogen atmosphere for 24 hours. The resultant reaction mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to yield the title compound (0.928 g, 95%).

¹H-NMR (CDCl₃) δ: 7.99 (1H, d, J=2.4 Hz), 7.38 (1H, dd, J=8.8, 2.4 Hz), 6.53 (1H, d, J=8.8 Hz), 4.50 (2H, brs), 4.24 (2H, s), 3.78 (2H, t, J=5.1 Hz), 3.67 (2H, t, J=5.4 Hz), 1.50 (9H, s).

Intermediates D-1 to D-41 were each synthesized by the process of Reference Example 13 and/or 14 with the corresponding halopyridine derivatives and amide derivatives. Appropriate protection or deprotection was performed as needed.

Reference Example 15 Synthesis of dimethyl-[2-(6-nitropyridin-3-yloxy)ethyl]amine

2-Dimethylaminoethanol (0.32 mL, 3.17 mmol) was dissolved in DMF (4 mL) and cesium carbonate (1.03 g, 3.17 mmol) was added thereto, and the resultant suspension was stirred at room temperature for 10 minutes. 5-Fluoro-2-nitropyridine (0.30 g, 2.11 mmol) was added to the suspension at room temperature, and the mixture then was stirred at 80° C. for 16 hours. The reaction was monitored by LC/MS. After completion of the reaction, the reaction was quenched through addition of ice water, and the reaction mixture was extracted with ethyl acetate. The resultant organic phase was dried over anhydrous sodium sulfate, and the solid was separated by filtration. The filtrate was then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to yield the title compound (0.40 g, 90%).

Reference Example 16 Synthesis of 5-(2-dimethylaminoethoxy)pyridin-2-ylamine

Dimethyl-[2-(6-nitropyridin-3-yloxy)ethyl]amine synthesized in Reference Example 15 (0.40 g, 1.90 mmol) was dissolved in THF (5 mL) and ethanol (5 mL), and palladium-carbon (80 mg) was added to the solution. The mixture was stirred under a hydrogen atmosphere overnight. The resultant reaction mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure. The resultant crude product was washed with a solvent mixture of ethyl acetate and hexane (1:9) to yield the title compound (0.28 g, 82%).

Intermediates E-1 to E-61 were each synthesized by the process of Reference Examples 15 and/or 16 with the corresponding halopyridine derivatives, alcohol derivatives, or thiol derivatives. Appropriate protection or deprotection was performed as needed.

Reference Example 17 Synthesis of tert-butyl 4-(6-chloropyridazin-3-yl)piperazine-1-carboxylate

A solution of 3,6-dichloropyridazine (5.01 g, 33.6 mmol) and tert-butyl piperazine-1-carboxylate (6.88 g, 37.0 mmol) in DMF (50 mL) was added triethylamine (11.7 mL, 50.4 mmol) and stirred at 80° C. overnight. The resultant reaction mixture was cooled to room temperature, and water was added to the mixture. The mixture was extracted three times with a solvent mixture of dichloromethane and methanol (95:5) (50 mL). The resultant organic phases were combined together and dried over anhydrous magnesium sulfate. The resultant solid was separated by filtration, and the filtrate was then concentrated under reduced pressure. The resultant crude product was washed with diethyl ether to yield the title compound (7.0 g, 70%).

Reference Example 18 Synthesis of tert-butyl 4-(6-((diphenylmethylene)amino)pyridazin-3-yl)piperazine-1-carboxylate

Tert-butyl 4-(6-chloropyridazin-3-yl)piperazine-1-carboxylate synthesized in Reference Example 17 (59.8 mg, 0.20 mmol, benzophenone imine (43.5 mg, 0.24 mmol), tris(dibenzylideneacetone)dipalladium (9.2 mg, 0.010 mmol), BINAP (12.5 mg, 0.020 mmol), and cesium carbonate (130.3 mg, 0.40 mmol) were suspended in toluene (1.0 mL), and the suspension was stirred at 100° C. overnight. The resultant reaction mixture was cooled to room temperature and then filtered through Celite, and the Celite was washed with ethyl acetate. The filtrate was washed with saturated brine and dried over anhydrous magnesium sulfate, and the solid was separated by filtration. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to yield the title compound (67 mg, 76%).

Reference Example 19 Synthesis of tert-butyl 4-(6-aminopyridazin-3-yl)piperazine-1-carboxylate

Tert-butyl 4-(6-((diphenylmethylene)amino)pyridazin-3-yl)piperazine-1-carboxylate synthesized in Reference Example 18 (67 mg, 0.151 mmol) was dissolved in THF (0.76 mL). To the solution was added an aqueous citric acid solution (0.378 mL, 0.755 mmol, 2 mol/L) and the mixture was stirred at room temperature overnight. The resultant reaction mixture was neutralized with saturated aqueous sodium hydrogen carbonate solution (5 mL), and the mixture was extracted twice with ethyl acetate (5 mL). The resultant organic phases were combined together and dried over anhydrous magnesium sulfate, and the resultant solid was separated by filtration. The filtrate was concentrated under reduced pressure, and the resultant crude product was washed with tert-butyl methyl ether (5 mL) to yield the title compound (30 mg, 71%).

Intermediates F-1 to F-77 were each synthesized by any of the processes of Reference Examples 17 to 19 or a combination of the processes with the corresponding haloheteroaryl derivatives and amine derivatives. Appropriate protection or deprotection was performed as needed.

Intermediates G-1 to G-12 were each synthesized by any of the processes of Reference Examples 15, 18, and 19 or a combination of the processes with the corresponding halopyridazine, alcohol, or thiol derivative. Appropriate protection or deprotection was performed as needed.

Reference Example 20 Synthesis of tert-butyl 4-(6-nitropyridin-3-yl)piperidin-3-ene-1-carboxylate

3-Bromo-6-nitropyridine was reacted with tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydropyridine-1(2H)-carboxylate under heating in the presence of a palladium catalyst by using the process described in J. Med. Chem. 2010, 53, p. 7938-7957, to yield the title compound.

Reference Example 21 Synthesis of tert-butyl 4-(6-aminopyridin-3-yl)piperidine-1-carboxylate

Tert-butyl 4-(6-nitropyridin-3-yl)piperidin-3-ene-1-carboxylate synthesized in Reference Example 20 was reduced under a hydrogen atmosphere in the presence of palladium-carbon by using the process described in J. Med. Chem. 2010, 53, p. 7938-7957 to yield the title compound.

Intermediates H-1 to H-12 were each synthesized by the process of Reference Example 20 and/or 21 with the corresponding haloheteroaryl or boric acid derivative. Appropriate protection or deprotection was performed as needed.

Reference Example 22

Intermediate I-l was synthesized through the reaction of tert-butyl chlorosulfonylcarbamate with tert-butyl N-[5-(aminoethyl)-2-pyridyl]-N-tert-butoxycarbonylcarbamate synthesized by any of the processes of Reference Examples 8 to 10 or a combination of the processes, and then the removal of the Boc groups under acidic conditions.

Reference Example 23

Intermediate J-1 was synthesized through the reaction of potassium isocyanate with tert-butyl N-tert-butoxycarbonyl-N-[5-(N-methylaminoethyl)-2-pyridyl]carbamate synthesized as in Reference Example 8 to 10, and the removal of the Boc groups under acidic conditions.

Intermediate J-2 was synthesized through hydrogen reduction of the nitro group of 5-amino-2-nitropyridine in the presence of palladium hydroxide/activated carbon by the process of Reference Example 23.

Reference Example 24

Intermediate K-1 was synthesized through the reaction of isocyanatoethane with tert-butyl N-[5-(aminoethyl)-2-pyridyl]-N-tert-butoxycarbonylcarbamate synthesized by any of the processes of Reference Examples 8 to 10 or a combination of the processes, and the removal of the Boc groups under acidic conditions.

Reference Example 25

Intermediate L-1 was synthesized through the reaction of 2-methoxyethyl bromide with tert-butyl N-[5-(aminoethyl)-2-pyridyl]-N-tert-butoxycarbonylcarbamate synthesized by any of the processes of Reference Examples 8 to 10 or a combination of the processes, the removal of the Boc groups under acidic conditions, and the selective protection of the secondary amino moiety with a Boc group as in Reference Example 11.

Reference Example 26

Intermediate M-1 was synthesized through the esterification of the carboxylic acid moiety of 2-(6-chloropyridin-3-yl)acetic acid, dimethylation of the carbonyl group at the α-position, reduction of the ester moiety with LAH, oxidation of the resultant alcohol moiety, reductive amination with methylamine, protection with a Boc group, amination of the 2-chloropyridine moiety in the presence of a Pd catalyst, and deprotection.

Reference Example 27

Intermediate N-1 was synthesized through the reaction of 5-bromo-2-nitropyridine with tert-butyl cyanoacetate under basic conditions, removal of the tert-butyl group and decarboxylation under acidic conditions, and reduction of the cyano group.

Reference Example 28

Intermediate O-1 was synthesized through the reaction of an alkyne derivative with imide derivative, subsequent reaction with 5-bromo-2-nitropyridine under Sonogashira coupling reaction conditions, and reduction with hydrogen in the presence of palladium hydroxide/activated carbon, involving protection and deprotection.

Reference Example 29

Intermediate P-1 was synthesized through acylation of 2-(6-nitropyridin-3-yl)ethylamine and reduction with hydrogen in the presence of palladium hydroxide/activated carbon.

Intermediates P-2 to P-17 were each synthesized by the process of Reference Example 29 with the corresponding amine derivative synthesized by, for example, any of the processes of Reference Examples 8 to 10 and the corresponding acylating agent, involving appropriate deprotection as needed. Appropriate acylation conditions were selected depending on the structure to be introduced. For example, an acid chloride or combination of a carboxylic acid and a condensing agent was used as the acylating agent in place of an acid anhydride.

Reference Example 30

Intermediate Q-1 was synthesized by mesylation of 2-(6-nitropyridin-3-yl)ethylamine and reduction with hydrogen in the presence of palladium hydroxide/activated carbon.

Intermediates Q-2 to Q-9 were each synthesized by the process of Reference Example 29 with the corresponding amine derivative synthesized by, for example, any of the processes of Reference Examples 8 to 10, involving appropriate deprotection as needed.

Reference Example 31

Intermediate R-1 was synthesized through the reaction of 5-bromo-2-nitropyridine with an alkyne derivative under Sonogashira coupling reaction conditions, protection and reduction with hydrogen in the presence of palladium hydroxide/activated carbon, involving protection and deprotection.

Intermediates R-2 to R-6 were each synthesized by the process of Reference Example 31; i.e., by the reactions 1) to 3) in Reference Example 31 with a halopyridine derivative and the corresponding terminal alkyne derivative.

Reference Example 32

Intermediate S-1 was synthesized through the mesylation of intermediate R-1 synthesized in Reference Example 31, reaction with an amide derivative under basic conditions and subsequent deprotection.

Intermediates S-2 and S-3 were each synthesized by the process of Reference Example 32 with the corresponding alcohol derivative, amide derivative, or sulfonamide derivative.

Reference Example 33

Intermediate T-1 was synthesized through basic hydrolysis of methyl 6-((tert-butoxycarbonyl)amino)nicotinate, condensation with morpholine, and deprotection.

Intermediates T-2 to T-17 were each synthesized by the process of Reference Example 33 with the corresponding ester derivative synthesized by, for example, the process of Reference Example 31, or the corresponding carboxylic acid derivative and amine derivative. Appropriate protection and deprotection were performed as needed.

Reference Example 34

Intermediate U-1 was synthesized by oxidation of 5-((3-((tert-butyldimethylsilyl)oxy)propyl)thio)-2-nitropyridine synthesized by the process of Reference Example 15 with m-chloroperbenzoic acid and reduction with hydrogen in the presence of palladium hydroxide/activated carbon.

Reference Example 35

Intermediate V-1 was synthesized through the reaction of 5-amino-2-nitropyridine with sodium azide and orthoformate and subsequent reduction with hydrogen in the presence of palladium hydroxide/activated carbon.

Reference Example 36

Intermediate W-1 was synthesized through the reaction of tert-butyl 2-chloro-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate with benzophenone imine and tert-butoxysodium in the presence of a Pd catalyst and deprotection.

Intermediates W-2 to W-4 were each synthesized by the process of Reference Example 36 with the corresponding halopyridine derivative.

Example 1 Synthesis of 3-(4-formyl-2-methylthiopyrimidin-5-yl)-2-propynyl benzoate

A solution of Pd(PhCN)₂Cl₂ (2.4 g, 6.4 mmol), copper iodide (0.82 g, 4.3 mmol), and [(t-Bu)₃P]HBF₄ (4 g, 13.9 mmol) in 1,4-dioxane (55 mL) was degassed and purged with argon, and diisopropylamine (18.5 mL, 128.8 mmol) was added to the solution at room temperature. The resultant reaction mixture was stirred at room temperature for five minutes. A solution of a mixture (25 g, crude product) of 5-bromo-2-methylsulfanylpyrimidine-4-carbaldehyde and (5-bromo-2-methylsulfanylpyrimidin-4-yl)methoxymethanol described in Reference Example 3 and propargyl benzoate (20 g, 128.8 mmol) in 1,4-dioxane (55 mL) was slowly added dropwise to the reaction mixture, and the reaction mixture was then stirred at room temperature for five hours. The reaction was monitored by LC/MS. After completion of the reaction, the reaction mixture was diluted with ethyl acetate (1 L). The mixture was subjected to suction filtration through Celite, and the Celite was washed with ethyl acetate. The filtrate was concentrated under reduced pressure, and the resultant crude product was directly used for the subsequent reaction.

Example 2 Synthesis of 6-((benzoyloxy)methyl)-2-(methylthio)pyrido[3,4-d]pyrimidine 7-oxide

The crude product of 3-(4-formyl-2-methylthiopyrimidin-5-yl)-2-propynyl benzoate synthesized in Example 1 was dissolved in ethanol (500 mL), and hydroxylamine hydrochloride (8.3 g, 120 mmol) and sodium acetate (10 g, 120 mmol) were added to the solution at room temperature. The resultant reaction mixture was stirred at room temperature for six hours, and then diluted with ethanol (1 L). Potassium carbonate (27.8 g, 200 mmol) was added to the mixture, and the mixture was then stirred at 50° C. for three hours. The reaction was monitored by LC/MS. After completion of the reaction, the reaction mixture was subjected to suction filtration through Celite, and the Celite was washed with ethyl acetate. The filtrate was dried over anhydrous sodium sulfate, and solid was separated by filtration. The filtrate was concentrated under reduced pressure, and the resultant crude product was purified by silica gel column chromatography to yield the title compound (5.0 g, 16%).

Example 3 Synthesis of 8-chloro-2-methylthiopyrido[3,4-d]pyrimidin-6-yl benzoate

The 6-((benzoyloxy)methyl)-2-(methylthio)pyrido[3,4-d]pyrimidine 7-oxide synthesized in Example 2 (5.0 g, 15.3 mmol) was dissolved in dichloromethane (60 mL) and cooled to 0° C. Thionyl chloride (25 mL, 343 mmol) was added dropwise to the solution at 0° C., and the mixture was stirred at room temperature for 16 hours. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was concentrated under reduced pressure, followed by azeotropic distillation twice with toluene (20 mL), to remove thionyl chloride. The residue was roughly purified by neutral alumina column chromatography to yield the title compound (2.75 g, 52%).

Example 4 Synthesis of (R)-1-(2-(methylthio)-8-(((S)-tetrahydro-2H-pyran-3-yl)amino)pyrido[3,4-d]pyrimidin-6-yl)ethyl benzoate

A mixture of (R)-1-(8-chloro-2-(methylthio)pyrido[3,4-d]pyrimidin-6-yl)ethyl benzoate synthesized by the process described in Example 3 (360 mg, 1.0 mmol), (S)-tetrahydro-2H-pyran-3-amine hydrochloride (206 mg, 1.5 mmol), and potassium carbonate (415 mg, 3.0 mmol) in 1,4-dioxane (4.0 mL) was stirred at 100° C. overnight. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was cooled to room temperature. The reaction mixture was diluted with water, and the mixture was extracted twice with ethyl acetate (10 mL). The resultant organic phase was washed with brine and dried over anhydrous magnesium sulfate. The solid was separated by filtration, and the filtrate was concentrated under reduced pressure. The resultant crude product was purified by silica gel column chromatography to yield the title compound (232 mg, 55%).

¹H-NMR (CDCl₃) δ: 8.97 (1H, s), 8.17-8.14 (2H, m), 7.62-7.57 (1H, m), 7.51-7.46 (2H, m), 6.87 (1H, s), 6.65 (1H, d, J=7.8 Hz), 6.10 (1H, q, J=6.7 Hz), 4.39-4.31 (1H, m), 4.08-4.03 (1H, m), 3.82-3.76 (1H, m), 3.70-3.64 (1H, m), 3.56-3.51 (1H, m), 2.65 (3H, s), 2.09-2.02 (1H, m), 1.89-1.78 (2H, m), 1.76-1.65 (4H, m)

LC/MS: (M+H)⁺=425.2, C₂₂H₂₄N₄O₃S=424.16

Example 5 Synthesis of 2-methylthio-8-(propan-2-yl)aminopyrido[3,4-d]pyrimidin-6-ylmethanol

The 8-isopropylamino-2-methylthiopyrido[3,4-d]pyrimidine-6-yl benzoate synthesized by the process described in Example 4 (3.7 g, 10.0 mmol) was dissolved in methanol (20 mL) and THF (20 mL), and an aqueous solution (10 mL) of lithium hydroxide (0.96 g, 40 mmol) was added dropwise to the solution at room temperature. The resultant reaction mixture was stirred at room temperature for one hour. The reaction was monitored by LC/MS. After completion of the reaction, hydrochloric acid (2 mol/L) was added dropwise to the reaction mixture, to adjust the pH of the mixture to 7. The resultant solid was separated by filtration and dried under reduced pressure to yield the title compound (2.55 g, 96%).

Example 6 Synthesis of 2-methylthio-8-(propan-2-yl)aminopyrido[3,4-d]pyrimidine-6-carbaldehyde

The 2-methylthio-8-(propan-2-yl)aminopyrido[3,4-d]pyrimidin-6-ylmethanol synthesized in Example 5 (3.1 g, 11.7 mmol) was dissolved in dichloromethane (30 mL) and the solution was stirred at 0° C. Dess-Martin Periodinane (15 g, 35.2 mmol) was gradually added to the solution at 0° C., and the reaction mixture was stirred at room temperature for three hours. The reaction was monitored by LC/MS. After completion of the reaction, the reaction was quenched by addition of an aqueous sodium thiosulfate solution for reduction of excess reagent. The aqueous phase was extracted three times with dichloromethane (50 mL). The resultant organic phases were combined together and dried over anhydrous sodium sulfate. The solid was separated by filtration, and the filtrate was then concentrated under reduced pressure. The residue was purified by silica gel column chromatography to yield the title compound (2.9 g, 94%).

Example 7 Synthesis of 6-difluoromethyl-2-methylthio-N-(propan-2-yl)pyrido[3,4-d]pyrimidine-8-amine

The 2-methylthio-8-(propan-2-yl)aminopyrido[3,4-d]pyrimidine-6-carbaldehyde synthesized in Example 6 (2.9 g, 11.1 mmol) was dissolved in dichloromethane (30 mL) and the solution was stirred at 0° C. DAST (7.1 g, 44.2 mmol) was gradually added to the solution at 0° C., and the reaction mixture was stirred at room temperature for three hours. The reaction was monitored by LC/MS. After completion of the reaction, the reaction was quenched by addition of saturated aqueous sodium carbonate solution (20 mL). The aqueous phase was extracted three times with dichloromethane (50 mL). The resultant organic phases were combined together and dried over anhydrous sodium sulfate. The solid was separated by filtration, and the filtrate was then concentrated under reduced pressure. The residue was purified by silica gel column chromatography to yield the title compound (2.37 g, 75%).

Compounds Int-1 to Int-8 were synthesized by the process described in Example 4 or Examples 5 to 7 in an appropriate order depending on the substituents.

TABLE 1 Compound Exact No. Structure NMR (M + H) ⁺ Mass Int-1

1H-NMR (CDCl3) δ: 8.99 (1H, s), 8.17- 8.13 (2H, m), 7.63- 7.56 (1H, m), 7.52- 7.45 (2H, m), 6.90 (1H, s), 6.59 (1H, d, J = 6.3 Hz), 6.11 (1H, q, J = 6.7 Hz), 4.84-4.74 (1H, m), 4.11-4.00 (2H, m), 3.93-3.79 (2H, m), 2.64 (3H, s), 2.47- 2.35 (1H, m), 2.06- 1.95 (1H, m), 1.73 (3H, d, J = 6.8 Hz). Int-2

1H-NMR (CDCl3) δ: 8.98 (1H, s), 8.18- 8.12 (2H, m), 7.63- 7.56 (1H, m), 7.52- 7.44 (2H, m), 6.87 (1H, s), 6.76-6.68 (1H, br m), 6.15- 6.06 (1H, m), 3.98- 3.55 (6H, m), 2.80- 2.62 (4H, m), 2.12- 2.01 (1H, m), 1.79- 1.69 (4H, m). Int-3

1H-NMR (CDCl3) δ: 9.01 (1H, s), 6.87 (1H, s), 6.42 (1H, d, J = 7.3 Hz), 4.39-4.27 (2H, m), 4.07-4.00 (2H, m), 3.66-3.57 (2H, m), 3.40 (3H, s), 2.66 (3H, s), 2.18-2.09 (2H, m), 1.73-1.59 (2H, m), 1.48 (3H, d, J = 6.8 Hz). Int-4

453.3 452.19 Int-5

453.3 452.19 Int-6

383.10 382.15 Int-7

307.15 306.15 Int-8

384.10 383.13

Example 8 Synthesis of (R)-1-(2-(methylsulfonyl)-8-(((S)-tetrahydro-2H-pyran-3-yl)amino)pyrido[3,4-d]pyrimidin-6-yl)ethyl benzoate

The (R)-1-(2-(methylthio)-8-(((S)-tetrahydro-2H-pyran-3-yl)amino)pyrido[3,4-d]pyrimidin-6-yl)ethyl benzoate synthesized in Example 4 (232 mg, 0.55 mmol) and Oxone® (672 mg, 1.09 mmol) were added to THF (2.7 mL) and water (2.7 mL) and the reaction mixture was stirred at room temperature overnight. The reaction was monitored by LC/MS. After completion of the reaction, saturated aqueous sodium hydrogen carbonate solution was slowly added to the reaction mixture, and the aqueous phase was extracted three times with ethyl acetate. The resultant organic phases were combined together and washed with saturated brine, and then dried over anhydrous magnesium sulfate. The solid was separated by filtration, and the filtrate was then concentrated under reduced pressure. The residue was purified by silica gel column chromatography to yield a crude product of the title compound (245 mg, 98%).

¹H-NMR (CDCl₃) δ: 9.30 (1H, s), 8.16 (2H, d, J=7.3 Hz), 7.65-7.60 (1H, m), 7.53-7.48 (2H, m), 7.02 (1H, s), 6.87 (1H, d, J=7.8 Hz), 6.13 (1H, q, J=6.7 Hz), 4.45-4.36 (1H, m), 4.08-4.04 (1H, m), 3.85-3.80 (1H, m), 3.67-3.60 (1H, m), 3.52-3.47 (1H, m), 3.41 (3H, s), 2.14-2.07 (1H, m), 1.90-1.74 (6H, m).

LC/MS: (M+H)⁺=457.2, C₂₂H₂₄N₄O₅S=456.15

Example 9 Synthesis of (R)-1-(8-(1-methoxy-2-methylpropan-2-ylamino)-2-(methylsulfinyl)pyrido[3,4-d]pyrimidin-6-yl)ethyl benzoate

(R)-1-(8-(l-methoxy-2-methylpropan-2-ylamino)-2-(methylthio)pyrido[3,4-d]pyrimidin-6-yl)ethyl benzoate synthesized by the process described in Example 7 (1.9 g, 4.46 mmol) was dissolved in dichloromethane (30 mL) and the solution was stirred at 0° C. m-CPBA (0.767 g, 4.46 mmol) was gradually added to the solution at 0° C., and the reaction mixture was stirred at room temperature overnight. The reaction was monitored by LC/MS. After completion of the reaction, the reaction was quenched by addition of an aqueous sodium thiosulfate solution for reduction of excess reagent. The aqueous phase was extracted three times with dichloromethane (30 mL). The resultant organic phases were combined together and washed once with saturated aqueous sodium hydrogen carbonate solution (50 mL) and once with saturated brine (50 mL). The organic phase was dried over anhydrous sodium sulfate, and the solid was separated by filtration. The filtrate was then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to yield the title compound (1.9 g, 96%).

Compounds Int-9 to Int-16 were synthesized by the process described in Example 8 or 9.

TABLE 2 Compound Exact No. Structure (M + H) ⁺ Mass Int-9

485.3 484.18 Int-10

485.3 484.18 Int-11

317.10 316.08 Int-12

459.15 458.16 Int-13

325.10 324.13 Int-14

415.10 414.14 Int-15

416.05 374.05 415.12 Int-16

339.15 338.14

Example 10 Synthesis of (R)-1-(5-chloro-2-(methylsulfonyl)-8-(((S)-tetrahydro-2H-pyran-3-yl)amino)pyrido[3,4-d]pyrimidin-6-yl)ethyl benzoate

A mixture of (R)-1-(2-(methylsulfonyl)-8-(((S)-tetrahydro-2H-pyran-3-yl)amino)pyrido[3,4-d]pyrimidin-6-yl)ethyl benzoate synthesized in Example 8 (268 mg, 0.587 mmol) and N-chlorosuccinimide (96 mg, 0.72 mmol) in 1,2-dichloroethane (2.9 mL) was stirred at 65° C. overnight. The reaction was monitored by LC/MS. After completion of the reaction, the reaction mixture was cooled to room temperature. The reaction mixture was directly purified by silica gel column chromatography to yield the title compound (255 mg, 89%).

¹H-NMR (CDCl₃) δ: 9.70 (1H, s), 8.11-8.06 (2H, m), 7.60-7.53 (1H, m), 7.48-7.42 (2H, m), 6.90 (1H, d, J=7.8 Hz), 6.46 (1H, q, J=6.7 Hz), 4.28-4.18 (1H, m), 3.82 (1H, dd, J=11.5, 3.2 Hz), 3.76-3.69 (1H, m), 3.65-3.56 (1H, m), 3.45-3.37 (4H, m), 2.09-2.00 (1H, m), 1.88-1.61 (6H, m).

Compound Int-17 was synthesized by the process described in Example 10.

TABLE 3 Compound Exact No. Structure (M + H) ⁺ Mass Int-17

449.10 448.10

Example 11 Synthesis of tert-butyl 4-[6-(6-difluoromethyl-8-isopropylaminopyrido[3,4-d]pyrimidin-2-ylamino)pyridin-3-yl]piperazine-1-carboxylate

The tert-butyl 4-(6-aminopyridin-3-yl)piperazine-1-carboxylate synthesized in Reference Example 5 (88 mg, 0.316 mmol) was dissolved in THF (3.5 mL), and sodium hydride (22.8 mg, 0.57 mmol, 60%) was added to the solution at 0° C. and stirred for 10 minutes. To the suspension was added a solution of the (6-difluoromethyl-2-methanesulfonylpyrido[3,4-d]pyrimidine-8-yl)isopropylamine synthesized in Example 8 (Int-11, 100 mg, 0.316 mmol) in THF (3.5 mL) at room temperature and the reaction mixture was stirred at 35° C. for one hour. The reaction was monitored by TLC and LC/MS. After completion of the reaction, the reaction was quenched by addition of ice water (10 mL). The aqueous phase was extracted twice with ethyl acetate (25 mL). The resultant organic phases were combined together and washed with saturated brine, and the mixture was dried over anhydrous sodium sulfate. The solid was separated by filtration, and the filtrate was then concentrated under reduced pressure. The residue was purified by silica gel column chromatography to yield the title compound (56.7 mg, 35%).

Example 12 Synthesis of 6-difluoromethyl-8-isopropyl-2-(5-piperazin-1-ylpyridin-2-yl)pyrido[3,4-d]pyrimidine-2,8-diamine (Compound 3)

The tert-butyl 4-[6-(6-difluoromethyl-8-isopropylaminopyrido[3,4-d]pyrimidin-2-ylamino)pyridin-3-yl]piperazine-1-carboxylate synthesized in Example 11 (195 mg, 0.378 mmol) was dissolved in dichloromethane (5 mL) and stirred at 0° C. Hydrogen chloride (0.4 mL, 4 mol/L, 1,4-dioxane solution) was added dropwise to the solution and stirred at room temperature for 30 minutes. The reaction was monitored by LC/MS. After completion of the reaction, the reaction mixture was concentrated under reduced pressure. The resultant crude product was purified by fractionation HPLC (acetonitrile/water/TFA) to yield a TFA salt of the title compound (114 mg, purity: 99% or more). The TFA salts obtained by multiple reactions were combined and used in the next step.

The TFA salt (200 mg) was dissolved in methanol (0.625 mL) and dichloromethane (1.875 mL) and applied onto a strong cation exchange resin (SCX) column. The SCX column was washed with a solvent mixture of methanol and dichloromethane (1:3). The target compound was subsequently eluted from the SCX column with a solvent mixture of methanol and dichloromethane (1:3) containing 2.5% ammonia (2 mol/L, methanol solution). The eluate was concentrated under reduced pressure to yield the title compound (105 mg, purity: >99%).

Example 13 Synthesis of tert-butyl (R)-4-(6-(6-(benzoyloxy)ethyl-8-(1-methoxy-2-methylpropan-2-ylamino)pyrido[3,4-d]pyrimidin-2-ylamino)pyridin-3-yl)piperazine-1-carboxylate

The (R)-1-(8-(1-methoxy-2-methylpropan-2-ylamino)-2-(methylsulfinyl)pyrido[3,4-d]pyrimidin-6-yl)ethyl benzoate synthesized in Example 9 (1.9 g, 4.3 mmol) and the tert-butyl 4-(6-aminopyridin-3-yl)piperazine-1-carboxylate synthesized in Reference Example 5 (3.59 g, 12.9 mmol) were suspended in toluene (30 mL) and the reaction mixture was stirred at 120° C. overnight. The reaction was monitored by LC/MS. The resultant reaction mixture was cooled to room temperature, and the solvent was removed through evaporation under reduced pressure. The residue was purified by silica gel column chromatography to yield the title compound (850 mg, 30%).

Example 14 Synthesis of (R)-1-(8-(l-methoxy-2-methylpropan-2-ylamino)-2-(5-(piperazin-1-yl)pyridin-2-ylamino)pyrido[3,4-d]pyrimidin-6-yl)ethanol (Compound 195)

The tert-butyl (R)-4-(6-(6-(benzoyloxy)ethyl-8-(1-methoxy-2-methylpropan-2-ylamino)pyrido[3,4-d]pyrimidin-2-ylamino)pyridin-3-yl)piperazine-1-carboxylate synthesized in Example 13 (850 mg, 1.3 mmol) was dissolved in THF (15 mL) and methanol (15 mL), and lithium hydroxide (124 mg, 5.2 mmol) was added to the solution. The resultant reaction mixture was stirred at room temperature overnight, and the reaction was monitored by LC/MS. After completion of the reaction, hydrogen chloride (4 mol/L, methanol solution) was added dropwise to the reaction mixture, to adjust the pH of the mixture to 7. The reaction mixture was concentrated under reduced pressure to yield a crude product. The crude product was used for the subsequent reaction without purification.

The crude product was dissolved in hydrogen chloride (20 mL, 4 mol/L, methanol solution) and stirred at room temperature for four hours. The reaction was monitored by LC/MS. After completion of the reaction, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in methanol (30 mL), and concentrated aqueous ammonia (25%) was added dropwise to the solution, to adjust the pH of the solution to 10 or higher. Saturated brine (100 mL) was added to the solution, and the mixture was extracted three times with a solvent mixture of dichloromethane and methanol (9:1) (30 mL). The resultant organic phases were combined together and washed once with saturated brine (50 mL). The organic phase was dried over anhydrous sodium sulfate, and the solid was separated by filtration. The filtrate was concentrated under reduced pressure to yield a crude product of the title compound. The crude product was then washed with methanol to yield the title compound (470 mg, 80%).

Example 15 Synthesis of (S)-1-(4-(6-((6-((R)-1-hydroxyethyl)-8-(isopropylamino)pyrido[3,4-d]pyrimidin-2-yl)amino)pyridazin-3-yl)piperazin-1-yl)propan-2-ol (Compound 676)

(R)-1-(8-(isopropylamino)-2-((6-(piperazin-1-yl)pyridazin-3-yl)amino)pyrido[3,4-d]pyrimidin-6-yl)ethanol synthesized by the process described in Example 14 (compound 261, 25 mg, 0.061 mmol) was dissolved in methanol (0.31 mL), and (S)-propylene oxide (3.5 mg, 0.061 mmol) was added to the solution. The resultant reaction mixture was stirred at 55° C. overnight, and the reaction was monitored by LC/MS. After completion of the reaction, the reaction mixture was concentrated under reduced pressure. The resultant crude product was purified by fractionation HPLC (acetonitrile/water/TFA) and applied onto a strong cation exchange resin (SCX) column. The SCX column was washed with methanol, and the target product was then eluted with ammonia (2 mol/L, methanol solution). The eluate was concentrated under reduced pressure to yield the title compound (19 mg).

Example 16 Synthesis of (R)-1-(8-(isopropylamino)-2-((6-(4-(oxetan-3-yl)piperazin-1-yl)pyridazin-3-yl)amino)pyrido[3,4-d]pyrimidin-6-yl)ethanol (Compound 682)

(R)-1-(8-(isopropylamino)-2-((6-(piperazin-1-yl)pyridazin-3-yl)amino)pyrido[3,4-d]pyrimidin-6-yl)ethanol synthesized by the process described in Example 14 (compound 261, 16.4 mg, 0.040 mmol) was dissolved in acetic acid (2.8 μL) and 1,2-dichloroethane (0.4 mL). To the mixture was added 3-oxetanone (2.8 μL, 0.048 mmol) and sodium triacetoxyborohydride (12.7 mg, 0.060 mmol). The resultant reaction mixture was stirred at 55° C. for two hours, and the reaction was monitored by LC/MS. After completion of the reaction, the reaction mixture was cooled to room temperature, and the reaction was quenched by addition of water. The reaction mixture was extracted with ethyl acetate, and the organic layer was concentrated under reduced pressure. The resultant crude product was then purified by amine-modified column chromatography (ethyl acetate/methanol) to yield the title compound (7.5 mg).

Example 17 Synthesis of (R)-3-(4-(6-((8-(isopropylamino)-6-(l-methoxyethyl)pyrido[3,4-d]pyrimidin-2-yl)amino)pyridazin-3-yl)piperazin-1-yl)propanoic acid (Compound 684)

(R)—N8-isopropyl-6-(1-methoxyethyl)-N2-(6-(piperazin-1-yl)pyridazin-3-yl)pyrido[3,4-d]pyrimidine-2,8-diamine synthesized by the process described in Example 14 (compound 217, 29.6 mg, 0.07 mmol) was dissolved in methanol (0.35 mL), and methyl acrylate (6.3 μL, 0.07 mmol) was added to the solution. The resultant reaction mixture was stirred at 55° C. for two hours, and the reaction was monitored by LC/MS. After completion of the reaction, the reaction mixture was concentrated under reduced pressure, and the resultant crude product was roughly purified by silica gel column chromatography (ethyl acetate/heptane). The crude product was then dissolved in THF (0.56 mL) and methanol (0.56 mL), and 4M aqueous lithium hydroxide solution (0.028 mL, 0.112 mmol) was added to the solution. The resultant reaction mixture was stirred at room temperature overnight, and the reaction was monitored by LC/MS. After completion of the reaction, the reaction mixture was acidified with 2M aqueous hydrochloric acid solution and then adsorbed onto a strong cation exchange resin (SCX) column. The SCX column was washed with water and dichloromethane, and the target product was then eluted with ammonia (2 mol/L, methanol solution). The eluate was concentrated under reduced pressure to yield the title compound (27.5 mg).

Example 18 Synthesis of (R)-2-(4-(6-((8-(isopropylamino)-6-(1-methoxyethyl)pyrido[3,4-d]pyrimidin-2-yl)amino)pyridazin-3-yl)piperazin-1-yl)-2-methylpropanoic acid (Compound 678)

(R)—N8-isopropyl-6-(1-methoxyethyl)-N2-(6-(piperazin-1-yl)pyridazin-3-yl)pyrido[3,4-d]pyrimidine-2,8-diamine synthesized by the process described in Example 14 (compound 217, 42.3 mg, 0.10 mmol) was dissolved in acetonitrile (0.2 mL). To the mixture was added tert-butyl 2-bromo-2-methylpropanoate (22.4 μL, 0.12 mmol) and potassium carbonate (16.6 mg). The resultant reaction mixture was stirred at 85° C. overnight, and the reaction was monitored by LC/MS. After completion of the reaction, the reaction mixture was cooled to room temperature, and the reaction was quenched by addition of water. The reaction mixture was extracted with ethyl acetate, and the resultant crude product was briefly purified by silica gel column chromatography (ethyl acetate/heptane). The crude product was then dissolved in dichloromethane (1 mL), and trifluoroacetic acid (1 mL) was added to the solution. The resultant reaction mixture was stirred at room temperature for 24 hours, and the reaction was monitored by LC/MS. After completion of the reaction, the reaction mixture was concentrated under reduced pressure, and the resultant crude product was adsorbed onto a strong cation exchange resin (SCX) column. The SCX column was washed with methanol, and the target product was then eluted with ammonia (2 mol/L, methanol solution). The eluate was concentrated under reduced pressure to yield the title compound (8.5 mg).

Example 19

Compounds 1 to 1239 were synthesized by the processes described in Examples 11 to 18.

TABLE 4 Com- pound No. L R¹ R² R³

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

92

93

94

95

96

97

98

99

100

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

151

152

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174

175

176

177

178

179

180

181

182

183

184

185

186

187

188

189

190

191

192

193

194

195

196

197

198

199

200

201

202

203

204

205

206

207

208

209

210

211

212

213

214

215

216

217

218

219

220

221

222

223

224

225

226

227

228

229

230

231

232

233

234

235

236

237

238

239

240

241

242

243

244

245

246

247

248

249

250

251

252

253

254

255

256

257

258

259

260

261

262

263

264

265

266

267

268

269

270

271

272

273

274

275

276

277

278

279

280

281

282

283

284

285

286

287

288

289

290

291

292

293

294

295

296

297

298

299

300

301

302

303

304

305

306

307

308

309

310

311

312

313

314

315

316

317

318

319

320

321

322

323

324

325

326

327

328

329

330

331

332

333

334

335

336

337

338

339

340

341

342

343

344

345

346

347

348

349

350

351

352

353

354

355

356

357

358

359

360

361

362

363

364

365

366

367

368

369

370

371

372

373

374

375

376

377

378

379

380

381

382

383

384

385

386

387

388

389

390

391

392

393

394

395

396

397

398

399

400

401

402

403

404

405

406

407

408

409

410

411

412

413

414

415

416

417

418

419

420

421

422

423

424

425

426

427

428

429

430

431

432

433

434

435

436

437

438

439

440

441

442

443

444

445

446

447

448

449

450

451

452

453

454

455

456

457

458

459

460

461

462

463

464

465

466

467

468

469

470

471

472

473

474

475

476

477

478

479

480

481

482

483

484

485

486

487

488

489

490

491

492

493

494

495

496

497

498

499

500

501

502

503

504

505

506

507

508

509

510

511

512

513

514

515

516

517

518

519

520

521

522

523

524

525

526

527

528

529

530

531

532

533

534

535

536

537

538

539

540

541

542

543

544

545

546

547

548

549

550

551

552

553

554

555

556

557

558

559

560

561

562

563

564

565

566

567

568

569

570

571

572

573

574

575

576

577

578

579

580

581

*faster isomer

582

*later isomer

583

584

585

586

587

588

589

590

591

592

593

594

595

596

597

598

599

600

601

602

603

604

605

606

607

608

609

610

611

612

613

614

615

616

617

618

619

620

621

622

623

624

625

626

627

628

629

630

631

632

633

634

635

636

637

638

639

640

641

642

643

644

645

646

647

648

649

650

651

652

653

654

655

656

657

658

659

660

661

662

663

664

665

666

667

668

669

670

*faster isomer

672

*later isomer

673

674

675

676

677

678

679

680

681

682

683

684

685

686

687

688

689

*faster isomer

690

*later isomer

691

692

693

694

695

696

697

698

699

700

701

702

703

704

*later isomer

705

*faster isomer

706

707

708

709

710

711

712

713

*later isomer

714

*faster isomer

715

716

717

718

719

720

721

722

723

724

*faster isomer

725

*later isomer

726

727

728

729

*later isomer

730

*faster isomer

731

732

733

734

735

*faster isomer

736

*later isomer

737

738

739

740

741

742

743

744

745

746

747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

763

764

765

766

767

768

769

770

771

772

773

*faster isomer

774

*later isomer

775

776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

793

794

795

796

797

798

799

800

801

802

803

804

805

806

807

808

809

*faster isomer

810

*later isomer

811

812

813

814

815

816

817

818

819

820

821

822

823

824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

865

*faster isomer

866

*later isomer

867

868

869

*faster isomer

870

*later isomer

871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

886

887

888

889

890

891

892

893

894

895

896

897

898

899

900

901

902

903

904

905

906

907

908

909

910

911

912

913

914

915

916

917

918

919

920

921

922

923

924

925

926

927

928

929

930

931

932

933

934

935

936

937

938

*faster isomer

939

*later isomer

940

*faster isomer

941

*later isomer

942

*faster isomer

943

944

945

946

947

948

949

950

951

952

953

954

955

956

957

958

959

960

961

962

963

964

965

966

967

968

969

970

971

972

973

974

975

976

977

978

979

980

981

982

983

984

985

986

987

988

989

990

991

992

*faster isomer

993

*later isomer

994

995

996

997

998

999

1000

1001

1002

1003

1004

1005

1006

1007

1008

1009

1010

1011

1012

1013

1014

1015

1016

1017

1018

1019

1020

1021

1022

1023

1024

1025

1026

1027

1028

1029

1030

1031

1032

1033

1034

*faster isomer

1035

*later isomer

1036

*faster isomer

1037

*later isomer

1038

1039

1040

1041

1042

1043

1044

1045

1046

1047

1048

1049

1050

1051

1052

1053

1054

1055

1056

1057

1058

1059

1060

1061

1062

1063

1064

1065

1066

*faster isomer

1067

*later isomer

1068

*faster isomer

1069

*later isomer

1070

1071

1072

1073

1074

1075

1076

1077

1078

1079

1080

1081

1082

1083

1084

1085

1086

1087

1088

*faster isomer

1089

*later isomer

1090

1091

1092

1093

1094

1095

1096

1097

1098

1099

1100

1101

1102

1103

1104

1105

1106

1107

1108

1109

1110

1111

1112

1113

1114

1115

1116

1117

1118

1119

1120

1121

1122

1123

1124

1125

1126

1127

1128

1129

1130

1131

1132

1133

1134

1135

1136

1137

1138

1139

1140

1141

1142

1143

1144

1145

1146

1147

1148

1149

1150

1151

1152

1153

1154

1155

1156

1157

1158

1159

1160

1161

1162

1163

1164

1165

1166

1167

1168

1169

1170

1171

1172

1173

1174

1175

1176

1177

1178

1179

1180

1181

1182

1183

1184

1185

1186

1187

1188

1189

1190

1191

1192

1193

1194

1195

1196

1197

1198

1199

1200

1201

1202

1203

1204

1205

1206

*faster isomer

1207

*later isomer

1208

1209

1210

1211

1212

1213

1214

1215

1216

1217

1218

1219

1220

1221

1222

1223

1224

1225

1226

1227

1228

1229

1230

1231

1232

1233

1234

1235

1236

1237

1238

1239

-   -   *: The compounds separated as optically active isomers by HPLC         purification with a chiral column (note: absolute configuration         is not determined).

Abbreviations

-   -   DEA: diethylamine, TEA=triethylamine, DCM: dichloromethane, IPA:         isopropylalcohol     -   (1) Compound 581: DAICEL CHIRALPAK AD 5 μm, n-hexane/MeOH/IPA,         RT=12.31 min.: optically active isomer with shorter retention         time.     -   (1) Compound 582: DAICEL CHIRALPAK AD 5 μm, n-hexane/MeOH/IPA,         RT=15.35 min.: optically active isomer with longer retention         time.     -   (2) Compound 671: CHIRALPAK IA-3 μm, 100% EtOH, RT=4.388 min.:         optically active isomer with shorter retention time.     -   (2) Compound 672: CHIRALPAK IA-3 μm, 100% EtOH, RT=6.490 min.:         optically active isomer with longer retention time.     -   (3) Compound 689: DAICEL CHIRALPAK AD-H 5 μm, n-hexane/EtOH,         RT=17.61 min.: optically active isomer with shorter retention         time.     -   (3) Compound 690: DAICEL CHIRALPAK AD-H 5 μm, n-hexane/EtOH,         RT=20.71 min.: optically active isomer with longer retention         time.     -   (4) Compound 704: CHIRALPAK AD-3 μm, Hexane (0.2%         TEA)/EtOH=50:50, RT=6.446 min.: optically active isomer with         longer retention time.     -   (4) Compound 705: CHIRALPAK AD-3 μm, Hexane (0.2%         TEA)/EtOH=50:50, RT=4.679 min.: optically active isomer with         shorter retention time.     -   (5) Compound 713: CHIRALPAK IA-3 μm, 100% MeOH (0.1% DEA),         RT=3.628 min.: optically active isomer with longer retention         time.     -   (5) Compound 714: CHIRALPAK IA-3 μm, 100% MeOH (0.1% DEA),         RT=2.533 min.: optically active isomer with shorter retention         time.     -   (6) Compound 724: CHIRALPAK AD-3, Hexane (0.1% DEA)/EtOH=50:50,         RT=4.32 min.: optically active isomer with shorter retention         time.     -   (6) Compound 725: CHIRALPAK AD-3, Hexane (0.1% DEA)/EtOH=50:50,         RT=5.06 min.: optically active isomer with longer retention         time.     -   (7) Compound 729: CHIRALPAK IA-3, 0.46*5 cm; 3 μm, Hexane (0.1%         IPA)/EtOH=50:50, RT=4.01 min.: optically active isomer with         longer retention time.     -   (7) Compound 730: CHIRALPAK IA-3, 0.46*5 cm; 3 μm, Hexane (0.1%         IPA)/EtOH=50:50, RT=1.68 min.: optically active isomer with         shorter retention time.     -   (8) Compound 735: DAICEL CHIRALPAK AD-H 5 μm, Hexane/IPA=90/10,         RT=9.18 min.: optically active isomer with shorter retention         time.     -   (8) Compound 736: DAICEL CHIRALPAK AD-H 5 μm, Hexane/IPA=90/10,         RT=10.65 min.: optically active isomer with longer retention         time.     -   (9) Compound 773: DAICEL CHIRALPAK IA 5 μm, MeOH/IPA=6/4,         RT=17.90 min.: optically active isomer with shorter retention         time.     -   (9) Compound 774: DAICEL CHIRALPAK IA 5 μm, MeOH/IPA=6/4,         RT=21.84 min.: optically active isomer with longer retention         time.     -   (10) Compound 775: DAICEL CHIRALPAK IA 5 μm,         Hexane/MeOH/IPA=6/1/1, RT=10.25 min.: optically active isomer         with shorter retention time.     -   (10) Compound 776: DAICEL CHIRALPAK IA 5 μm,         Hexane/MeOH/IPA=6/1/1, RT=14.71 min.: optically active isomer         with longer retention time.     -   (11) Compound 809: DAICEL CHIRALPAK AD-H 5 μm, 4.6*250 mm,         n-Hexane/EtOH/MeOH=70/15/15, RT=21.62 min.: optically active         isomer with shorter retention time.     -   (11) Compound 810: DAICEL CHIRALPAK AD-H 5 μm, 4.6*250 mm,         n-Hexane/EtOH/MeOH=70/15/15, RT=25.87 min.: optically active         isomer with longer retention time.     -   (12) Compound 865: CHIRALPAK IA-3; 0.46*5 cm; 3 μm; Hexane (0.1%         DEA)/EtOH=70:30, RT=4.81 min.: optically active isomer with         shorter retention time.     -   (12) Compound 866: CHIRALPAK IA-3; 0.46*5 cm; 3 μm; Hexane (0.1%         DEA)/EtOH=70:30, RT=5.48 min.: optically active isomer with         longer retention time.     -   (13) Compound 869: CHIRALPAK IA-3; 0.46*5 cm; 3 μm; Hexane (0.1%         DEA)/EtOH=60:40, RT=2.27 min.: optically active isomer with         shorter retention time.     -   (13) Compound 870: CHIRALPAK IA-3; 0.46*5 cm; 3 μm; Hexane (0.1%         DEA)/EtOH=60:40, RT=2.93 min.: optically active isomer with         longer retention time.     -   (14) Compound 875: DAICEL CHIRALPAK AD-H 5 μm,         Hexane/MeOH/EtOH=80/10/10, RT=22.30 min.: optically active         isomer with shorter retention time.     -   (14) Compound 876: DAICEL CHIRALPAK AD-H 5 μm,         Hexane/MeOH/EtOH=80/10/10, RT=29.24 min.: optically active         isomer with longer retention time.     -   (15) Compound 938: DAICEL CHIRALPAK AD-H 5 μm, (100% EtOH+0.1%         DEA), RT=33.58 min.: optically active isomer with shorter         retention time.     -   (15) Compound 939: DAICEL CHIRALPAK AD-H 5 μm, (100% EtOH+0.1%         DEA), RT=42.68 min.: optically active isomer with longer         retention time.     -   (16) Compound 940: DAICEL CHIRALPAK AD-H 5 μm,         (Hexane/MeOH/EtOH+0.1% DEA=6/2/2), RT=20.23 min.: optically         active isomer with shorter retention time.     -   (16) Compound 941: DAICEL CHIRALPAK AD-H 5 μm,         (Hexane/MeOH/EtOH+0.1% DEA=6/2/2), RT=22.07 min.: optically         active isomer with longer retention time.     -   (17) Compound 942: DAICEL CHIRALPAK AD-H 5 μm,         (Hexane/MeOH/EtOH+0.1% DEA=6/2/2), RT=13.99 min.: optically         active isomer with shorter retention time.     -   (17) Compound 943: DAICEL CHIRALPAK AD-H 5 μm,         (Hexane/MeOH/EtOH+0.1% DEA=6/2/2), RT=15.66 min.: optically         active isomer with longer retention time.     -   (18) Compound 989: CHIRALPAK IA, n-Hexane/EtOH=70/30, RT=13.32         min.: optically active isomer with shorter retention time.     -   (18) Compound 990: CHIRALPAK IA, n-Hexane/EtOH=70/30, RT=16.69         min.: optically active isomer with longer retention time.     -   (19) Compound 992: CHIRALPAK IA-3: 0.46*5 cm; 3 μm, Hexane (0.1%         DEA)/EtOH=50:50, 1.5 ml/min, RT=1.87 min.: optically active         isomer with shorter retention time.     -   (19) Compound 993: CHIRALPAK IA-3: 0.46*5 cm; 3 μm, Hexane (0.1%         DEA)/EtOH=50:50, 1.5 ml/min, RT=3.56 min.: optically active         isomer with longer retention time.     -   (20) Compound 1025: CHIRALPAK IA, 0.46*25 cm; 5 n, MeOH (0.1%         DEA)/DCM=75:25, 1.0 mL/min., RT=6.597 min.: optically active         isomer with shorter retention time.     -   (20) Compound 1026: CHIRALPAK IA, 0.46*25 cm; 5 μm, MeOH (0.1%         DEA)/DCM=75:25, 1.0 mL/min., RT=8.199 min.: optically active         isomer with longer retention time.     -   (21) Compound 1034: CHIRALPAK AS-3, 0.46*10 cm; 3 nm, Hexane         (0.1% DEA)/(MeOH/EtOH=1:1)=70:30, 1.0 mL/min., RT=6.23 min.:         optically active isomer with shorter retention time.     -   (21) Compound 1035: CHIRALPAK AS-3, 0.46*10 cm; 3 μm, Hexane         (0.1% DEA)/(MeOH/EtOH=1:1)=70:30, 1.0 mL/min., RT=9.94 min.:         optically active isomer with longer retention time.     -   (22) Compound 1036: CHIRALPAK IC-3, 0.46*10 cm; 3 μm, DCM (0.1%         DEA)/MeOH=20:80, 1.0 mL/min., RT=5.10 min.: optically active         isomer with shorter retention time.     -   (22) Compound 1037: CHIRALPAK IC-3, 0.46*10 cm; 3 μm, DCM (0.1%         DEA)/MeOH=20:80, 1.0 mL/min., RT=5.95 min.: optically active         isomer with longer retention time.     -   (23) Compound 1066: CHIRALPAK IC-3, 0.46*10 cm; 3 μm, MeOH (0.1%         DEA)/DCM=95:5, 1.0 ml/min., RT=5.421 min.: optically active         isomer with shorter retention time.     -   (23) Compound 1067: CHIRALPAK IC-3, 0.46*10 cm; 3 μm, MeOH (0.1%         DEA)/DCM=95:5, 1.0 ml/min., RT=6.00 min.: optically active         isomer with longer retention time.     -   (24) Compound 1068: CHIRALPAK IC, 0.46*25 cm; 5 μm, MeOH (0.1%         DEA)/DCM=90:10, 1.0 ml/min., RT=17.429 min.: optically active         isomer with shorter retention time.     -   (24) Compound 1069: CHIRALPAK IC, 0.46*25 cm; 5 μm, MeOH (0.1%         DEA)/DCM=90:10, 1.0 ml/min., RT=20.57 min.: optically active         isomer with longer retention time.     -   (25) Compound 1088: CHIRALPAK IA, n-Hexane/EtOH/MeOH=50/25/25,         1.0 mL/min., RT=15.21 min.: optically active isomer with shorter         retention time.     -   (25) Compound 1089: CHIRALPAK IA, n-Hexane/EtOH/MeOH=50/25/25,         1.0 mL/min., RT=18.59 min.: optically active isomer with longer         retention time.     -   *: Compound separated as diastereomers by reversed phase HPLC         purification (note: absolute configuration is not determined).     -   (1) Compound 831: C-18: RT=1.67 min.: diastereomer with shorter         retention time.     -   (1) Compound 832: C-18: RT=1.71 min.: diastereomer with longer         retention time.     -   *: Compound represented its relative configurations of two         substituents.     -   Compound#: 577, 607, 608, 609, 610, 618, 619, 621, 622, 645,         646, 647, 673, 708, 739, 742, 799, 801, and 864.

Lengthy table referenced here US10124004-20181113-T00001 Please refer to the end of the specification for access instructions.

Example 20

Human CDK4/Cyclin D3 Inhibitory Activity

Each compound was analyzed for CDK4/cyclin D3 inhibitory activity with an assay kit (QS S Assist CDK4/Cyclin D3_FP Kit, available from Carna Biosciences, Inc.). This assay kit determines kinase activity on the basis of the IMAP technology by Molecular Devices. Specifically, the kinase activity is determined through quantification of a variation in fluorescent polarization caused by binding of a kinase-phosphorylated fluorescent substance to an IMAP-binding reagent.

Each solution was prepared with the 10× assay buffer attached to the kit or a separately prepared assay buffer having the same composition as the assay buffer attached to the kit. An assay buffer was prepared by 10-fold dilution of the 10× assay buffer with distilled water. The assay buffer contains 20 mM HEPES (pH 7.4), 0.01% Tween20, and 2 mM dithiothreitol. A test compound solution was prepared by dilution of the test compound with dimethyl sulfoxide (DMSO) to a concentration 100 times higher than the final concentration and then 25-fold dilution with the assay buffer to a concentration four times higher than the final concentration. An ATP/substrate/Metal solution was prepared by five-fold dilution of the 5×ATP/substrate/Metal solution attached to the kit with the assay buffer. An enzyme solution was prepared by dilution of the CDK4/cyclin D3 attached to the kit with the assay buffer to a concentration twice higher than the final concentration (final concentration of CDK4/cyclin D3:12.5 to 25 ng/well). A detection reagent was prepared by five-fold dilution of each of 5× IMAP-binding buffer A and 5×IMAP-binding buffer B with distilled water, mixing of IMAP-binding buffer A with IMAP-binding buffer B at a ratio of 85:15, and 400-fold dilution of the IMAP-binding reagent with the mixed buffer.

The test compound solution (5 μL/well) and the ATP/substrate/Metal solution (5 μL/well) were added to a 384-well plate, and the enzyme solution or the assay buffer (10 μL/well) was added to the plate (total amount of the reaction mixture: 20 μL/well) for initiation of enzymatic reaction. The reaction mixture had a composition of 20 mM HEPES (pH 7.4), 0.01% Tween 20, 2 mM dithiothreitol, 100 nM FITC-labeled peptide substrate (the sequence of the substrate peptide is not disclosed by Carna Biosciences, Inc.), 10 μM ATP, 1 mM magnesium chloride, 1% DMSO, and 12.5 to 25 ng/well CDK4/cyclin D3. The reaction was performed at room temperature for 45 minutes, and the detection reagent (60 μL/well) was then added to the plate, followed by further reaction for 30 minutes at room temperature under light shielding conditions. Subsequently, fluorescent polarization was determined with a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 535 nm.

The percent inhibition of enzyme activity was calculated for each test compound (note: enzyme activity=100% in the case of addition of the enzyme solution and addition of DMSO instead of the test compound solution, whereas enzyme activity=0% in the case of addition of the assay buffer instead of the enzyme solution, and addition of DMSO instead of the test compound solution). The percent inhibition of enzyme activity was fitted to a dose-response curve, to determine a 50% inhibitory concentration against CDK4/cyclin D3.

The inhibitory activity of each compound against CDK4/cyclin D3 was shown in tables described below.

In each table, “+++” corresponds to IC₅₀<10 nM, “++” 10 nM≤IC₅₀<100 nM, and “+” 100 nM≤IC₅₀.

Example 21

Human CDK2/Cyclin A² Inhibitory Activity

Each compound was analyzed for CDK2/cyclin A2 inhibitory activity with an assay kit (QS S Assist CDK2/Cyclin A2_FP Kit, available from Carna Biosciences, Inc.). This assay kit determines kinase activity on the basis of the IMAP technology by Molecular Devices. Specifically, the kinase activity is determined through quantification of a variation in fluorescent polarization caused by binding of a kinase-phosphorylated fluorescent substance to an IMAP-binding reagent.

An assay buffer was prepared by 10-fold dilution of the 10× assay buffer attached to the kit with distilled water, and each solution was prepared with the assay buffer. The assay buffer contained 20 mM HEPES (pH 7.4), 0.01% Tween 20, and 2 mM dithiothreitol. A test compound solution was prepared by dilution of the test compound with dimethyl sulfoxide (DMSO) to a concentration 100 times higher than the final concentration and then 25-fold dilution with the assay buffer to a concentration four times higher than the final concentration. An ATP/substrate/Metal solution was prepared by five-fold dilution of the 5×ATP/substrate/Metal solution attached to the kit with the assay buffer. An enzyme solution was prepared by dilution of the CDK2/cyclin A2 attached to the kit with the assay buffer to a concentration twice higher than the final concentration (final concentration of CDK2/cyclin A2: 2.5 ng/well). A detection reagent was prepared by five-fold dilution of 5×IMAP-binding buffer A with distilled water and 400-fold dilution of the IMAP-binding reagent with the diluted buffer.

The test compound solution (5 μL/well) and the ATP/substrate/Metal solution (5 μL/well) were added to a 384-well plate, and the enzyme solution or the assay buffer (10 μL/well) was added to the plate (total amount of the reaction mixture: 20 μL/well) for initiation of enzymatic reaction. The reaction mixture had a composition of 20 mM HEPES (pH 7.4), 0.01% Tween 20, 2 mM dithiothreitol, 100 nM FITC-labeled peptide substrate (the sequence of the substrate peptide is not disclosed by Carna Biosciences, Inc.), 30 μM ATP, 5 mM magnesium chloride, 1% DMSO, and 2.5 ng/well CDK2/cyclin A2. The reaction was performed at room temperature for 60 minutes, and the detection reagent (60 μL/well) was then added to the plate, followed by further reaction for 30 minutes at room temperature under light shielding conditions. Subsequently, fluorescent polarization was determined with a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 535 nm.

The percent inhibition of enzyme activity was calculated for each test compound (note: enzyme activity=100% in the case of addition of the enzyme solution and addition of DMSO instead of the test compound solution, whereas enzyme activity=0% in the case of addition of the assay buffer instead of the enzyme solution and addition of DMSO instead of the test compound solution). The percent inhibition of enzyme activity was fitted to a dose-response curve, to determine a 50% inhibitory concentration against CDK2/cyclin A2.

The inhibitory activity of each compound against CDK2/cyclin A2 was shown in tables described below.

In each table, “+++” corresponds to IC₅₀<10 nM, “++” 10 nM≤IC₅₀<100 nM, and “+” 100 nM≤IC₅₀.

TABLE 6 CDK4 CDK2 CDK4 CDK2 Compound Activity Activity Compound Activity Activity No. symbol symbol No. symbol symbol 1 +++ ++ 2 +++ ++ 3 +++ ++ 4 +++ ++ 5 +++ + 6 +++ ++ 7 +++ + 8 +++ + 9 +++ + 10 +++ + 11 ++ + 12 +++ + 13 +++ ++ 14 +++ + 15 +++ + 16 +++ + 17 +++ + 18 +++ + 19 +++ + 20 +++ + 21 +++ + 22 ++ + 23 +++ + 24 +++ + 25 + + 26 +++ + 27 ++ + 28 +++ + 29 +++ + 30 +++ ++ 31 +++ + 32 +++ + 33 + + 34 ++ + 35 +++ + 36 + + 37 +++ + 38 +++ + 39 +++ + 40 +++ + 41 +++ + 42 +++ ++ 43 +++ ++ 44 +++ + 45 +++ + 46 +++ ++ 47 +++ ++ 48 +++ + 49 +++ ++ 50 +++ ++ 51 ++ + 52 +++ ++ 53 +++ ++ 54 +++ + 55 +++ ++ 56 +++ + 57 +++ + 58 ++ + 59 +++ + 60 +++ + 61 +++ + 62 +++ + 63 +++ + 64 +++ + 65 ++ + 66 +++ + 67 +++ ++ 68 +++ + 69 +++ + 70 +++ + 71 +++ + 72 +++ + 73 +++ ++ 74 +++ + 75 +++ + 76 +++ + 77 +++ + 78 +++ + 79 +++ + 80 +++ ++ 81 +++ ++ 82 ++ + 83 +++ ++ 84 +++ ++ 85 ++ ++ 86 ++ + 87 +++ +++ 88 + + 89 +++ ++ 90 ++ ++ 91 +++ + 92 +++ + 93 +++ + 94 +++ + 95 + ++ 96 ++ ++ 97 ++ + 98 + + 99 + + 100 ++ + 101 +++ + 102 ++ + 103 +++ + 104 ++ + 105 +++ + 106 +++ + 107 ++ + 108 +++ ++ 109 ++ + 110 + + 111 ++ + 112 ++ ++ 113 +++ + 114 ++ + 115 +++ + 116 ++ + 117 +++ + 118 +++ ++ 119 ++ + 120 ++ + 121 +++ ++ 122 ++ + 123 ++ + 124 ++ + 125 +++ ++ 126 + + 127 +++ ++ 128 +++ ++ 129 +++ ++ 130 +++ ++ 131 +++ ++ 132 +++ ++ 133 ++ + 134 +++ ++ 135 +++ + 136 ++ + 137 +++ ++ 138 +++ ++ 139 +++ ++ 140 +++ + 141 +++ + 142 +++ ++ 143 ++ + 144 +++ + 145 +++ ++ 146 +++ + 147 +++ ++ 148 ++ + 149 ++ + 150 +++ ++ 151 +++ ++ 152 +++ ++ 153 +++ ++ 154 +++ + 155 +++ ++ 156 +++ + 157 ++ + 158 ++ + 159 +++ + 160 +++ 161 +++ ++ 162 ++ + 163 +++ + 164 +++ ++ 165 +++ + 166 +++ ++ 167 +++ + 168 +++ ++ 169 +++ ++ 170 +++ + 171 +++ ++ 172 ++ + 173 +++ ++ 174 +++ ++ 175 +++ ++ 176 ++ + 177 +++ ++ 178 +++ + 179 ++ + 180 ++ + 181 +++ ++ 182 ++ + 183 +++ ++ 184 +++ ++ 185 +++ ++ 186 ++ + 187 ++ + 188 ++ + 189 +++ ++ 190 +++ ++ 191 +++ ++ 192 ++ + 193 ++ + 194 +++ ++ 195 +++ + 196 +++ + 197 ++ + 198 ++ + 199 ++ + 200 +++ + 201 ++ + 202 ++ + 203 +++ ++ 204 +++ + 205 +++ ++ 206 +++ + 207 +++ + 208 +++ + 209 ++ + 210 ++ + 211 ++ ++ 212 +++ + 213 +++ ++ 214 +++ + 215 +++ + 216 ++ + 217 +++ + 218 +++ ++ 219 +++ ++ 220 +++ + 221 +++ + 222 ++ + 223 +++ + 224 +++ + 225 +++ + 226 +++ ++ 227 +++ + 228 ++ + 229 +++ + 230 +++ + 231 ++ + 232 +++ + 233 +++ + 234 +++ + 235 ++ + 236 +++ ++ 237 ++ + 238 +++ + 239 +++ + 240 +++ + 241 +++ + 242 ++ + 243 +++ + 244 +++ + 245 +++ + 246 +++ + 247 +++ + 248 ++ + 249 +++ + 250 +++ + 251 +++ + 252 +++ + 253 +++ + 254 +++ + 255 +++ + 256 +++ + 257 +++ + 258 +++ + 259 +++ + 260 +++ + 261 +++ + 262 ++ + 263 +++ + 264 +++ + 265 +++ + 266 +++ + 267 +++ + 268 ++ + 269 +++ + 270 +++ + 271 +++ + 272 +++ + 273 +++ + 274 +++ + 275 ++ + 276 +++ + 277 + + 278 +++ + 279 ++ + 280 + + 281 ++ + 282 +++ + 283 +++ + 284 +++ + 285 +++ + 286 +++ + 287 +++ + 288 +++ + 289 +++ + 290 +++ + 291 +++ + 292 ++ + 293 +++ + 294 +++ + 295 ++ + 296 + + 297 +++ + 298 ++ + 299 ++ + 300 +++ + 301 +++ 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411 +++ 412 +++ 413 +++ 414 ++ 415 +++ 416 ++ 417 +++ 418 +++ 419 ++ 420 +++ 421 ++ 422 ++ 423 +++ 424 +++ 425 +++ 426 +++ 427 +++ 428 +++ 429 +++ 430 +++ 431 +++ 432 +++ + 433 ++ 434 +++ 435 ++ 436 ++ 437 + 438 +++ 439 ++ 440 ++ 441 + 442 + 443 ++ 444 + 445 +++ 446 ++ 447 ++ 448 + 449 + 450 +++ 451 +++ 452 +++ 453 +++ 454 +++ 455 +++ 456 +++ + 457 +++ 458 +++ 459 +++ 460 +++ 461 ++ 462 ++ 463 +++ + 464 +++ 465 +++ 466 +++ + 467 +++ + 468 +++ 469 ++ 470 + 471 ++ 472 ++ 473 ++ 474 +++ + 475 ++ 476 +++ 477 +++ 478 +++ 479 +++ 480 ++ 481 ++ 482 +++ 483 +++ 484 +++ 485 +++ 486 +++ 487 +++ 488 +++ 489 +++ 490 +++ 491 +++ + 492 +++ ++ 493 +++ 494 +++ 495 +++ 496 +++ 497 +++ + 498 +++ 499 +++ ++ 500 +++ 501 +++ + 502 +++ 503 +++ 504 +++ 505 ++ 506 ++ 507 +++ + 508 +++ + 509 +++ + 510 +++ + 511 +++ ++ 512 +++ + 513 ++ 514 ++ 515 ++ 516 ++ 517 +++ 518 +++ 519 +++ + 520 +++ 521 +++ ++ 522 +++ ++ 523 +++ 524 +++ + 525 +++ 526 +++ + 527 ++ 528 +++ 529 +++ + 530 +++ 531 +++ 532 +++ + 533 +++ 534 +++ 535 +++ 536 +++ 537 +++ + 538 +++ 539 ++ 540 +++ 541 +++ 542 ++ 543 +++ 544 +++ 545 +++ 546 +++ 547 +++ 548 +++ + 549 +++ + 550 +++ + 551 ++ 552 +++ 553 +++ + 554 +++ + 555 +++ + 556 +++ ++ 557 +++ + 558 +++ ++ 559 +++ + 560 +++ + 561 +++ + 562 +++ + 563 +++ 564 +++ 565 +++ 566 +++ + 567 +++ + 568 +++ 569 +++ + 570 +++ 571 +++ 572 +++ + 573 +++ + 574 +++ + 575 +++ + 576 +++ + 577 +++ 578 +++ 579 +++ + 580 ++ 581 +++ + 582 +++ 583 +++ + 584 +++ + 585 +++ + 586 +++ + 587 +++ 588 +++ + 589 +++ + 590 +++ + 591 +++ + 592 +++ + 593 +++ + 594 +++ 595 +++ + 596 +++ + 597 +++ + 598 +++ 599 ++ 600 +++ + 601 +++ + 602 +++ + 603 +++ 604 +++ + 605 +++ ++ 606 +++ + 607 +++ 608 +++ + 609 +++ + 610 +++ + 611 +++ + 612 +++ ++ 613 ++ 614 +++ + 615 +++ + 616 +++ 617 +++ 618 +++ + 619 +++ + 620 +++ 621 +++ + 622 +++ + 623 +++ ++ 624 +++ + 625 +++ + 626 +++ + 627 +++ + 628 +++ + 629 +++ + 630 +++ + 631 +++ ++ 632 +++ + 633 +++ + 634 +++ + 635 +++ + 636 +++ + 637 +++ + 638 +++ + 639 +++ + 640 +++ + 641 +++ + 642 +++ + 643 +++ + 644 +++ + 645 +++ + 646 +++ + 647 +++ + 648 +++ + 649 +++ + 650 +++ + 651 +++ + 652 +++ + 653 +++ + 654 +++ + 655 ++ + 656 +++ + 657 +++ + 658 +++ + 659 +++ + 660 +++ + 661 +++ + 662 +++ + 663 +++ + 664 +++ + 665 +++ + 666 +++ + 667 +++ + 668 +++ + 669 ++ + 670 + + 672 +++ + 673 +++ + 674 +++ + 675 +++ + 676 +++ + 677 +++ + 678 ++ + 679 ++ + 680 ++ + 681 ++ + 682 +++ + 683 +++ + 684 ++ 685 ++ 686 +++ + 687 +++ 688 +++ 689 +++ 690 +++ 691 +++ + 692 +++ + 693 +++ + 694 ++ + 695 +++ + 696 +++ + 697 ++ 698 +++ + 699 ++ 700 +++ + 701 +++ + 702 ++ + 703 ++ + 704 +++ 705 +++ 706 +++ + 707 +++ 708 +++ + 709 +++ + 710 +++ + 711 +++ + 712 +++ + 713 +++ + 714 + + 715 ++ + 716 ++ + 717 ++ + 718 ++ + 719 +++ + 720 +++ + 721 +++ + 722 +++ + 723 +++ + 724 +++ + 725 +++ + 726 +++ + 727 +++ + 728 +++ + 729 +++ + 730 ++ + 731 +++ + 732 +++ + 733 ++ + 734 +++ + 735 +++ + 736 +++ + 737 +++ + 738 +++ + 739 +++ + 740 +++ + 741 +++ + 742 +++ + 743 +++ + 744 +++ + 745 +++ + 746 +++ + 747 ++ + 748 ++ + 749 +++ + 750 +++ + 751 +++ + 752 ++ + 753 +++ + 754 ++ + 755 +++ + 756 ++ + 757 +++ + 758 ++ + 759 ++ + 760 ++ + 761 +++ + 762 +++ + 763 +++ + 764 +++ + 765 +++ + 766 ++ + 767 +++ + 768 +++ + 769 +++ + 770 ++ + 771 +++ + 772 +++ + 773 +++ + 774 ++ + 775 +++ + 776 +++ + 777 +++ + 778 +++ + 779 +++ + 780 ++ + 781 +++ + 782 +++ + 783 +++ + 784 +++ + 785 +++ + 786 +++ + 787 +++ + 788 +++ + 789 +++ + 790 +++ + 791 +++ ++ 792 ++ + 793 ++ + 794 +++ + 795 +++ ++ 796 +++ + 797 ++ + 798 +++ + 799 +++ + 800 +++ + 801 +++ + 802 +++ + 803 +++ + 804 +++ + 805 +++ + 806 +++ + 807 ++ + 808 +++ + 809 +++ ++ 810 +++ + 811 +++ ++ 812 ++ + 813 +++ + 814 +++ + 815 +++ + 816 +++ + 817 +++ + 818 +++ + 819 +++ + 820 +++ ++ 821 +++ ++ 822 +++ ++ 823 ++ + 824 +++ + 825 +++ + 826 +++ + 827 +++ + 828 ++ + 829 +++ + 830 +++ + 831 ++ + 832 ++ + 833 +++ + 834 +++ + 835 ++ + 836 ++ + 837 +++ ++ 838 +++ + 839 +++ + 840 + + 841 +++ + 842 ++ + 843 +++ + 844 +++ + 845 +++ + 846 +++ + 847 +++ + 848 +++ ++ 849 +++ ++ 850 ++ ++ 851 ++ + 852 +++ + 853 +++ + 854 +++ + 855 +++ + 856 +++ + 857 +++ + 858 +++ + 859 +++ + 860 +++ + 861 +++ + 862 +++ + 863 +++ + 864 +++ + 865 +++ + 866 +++ + 867 +++ + 868 +++ + 869 +++ ++ 870 +++ + 871 ++ + 872 ++ + 873 +++ + 874 +++ + 875 +++ + 876 +++ + 877 +++ + 878 +++ + 879 +++ + 880 +++ + 881 +++ + 882 +++ + 883 +++ + 884 +++ + 885 +++ + 886 +++ + 887 +++ + 888 +++ + 889 ++ + 890 ++ + 891 ++ + 892 ++ + 893 ++ + 894 ++ + 895 +++ + 896 +++ + 897 ++ + 898 +++ + 899 +++ + 900 +++ + 901 +++ + 902 +++ + 903 +++ ++ 904 +++ + 905 +++ + 906 ++ + 907 +++ + 908 +++ + 909 +++ + 910 ++ + 911 +++ + 912 +++ + 913 +++ + 914 +++ + 915 +++ + 916 +++ + 917 +++ + 918 +++ + 919 +++ + 920 +++ + 921 +++ + 922 +++ + 923 +++ + 924 +++ + 925 +++ + 926 +++ + 927 +++ + 928 +++ + 929 +++ + 930 +++ + 931 +++ + 932 +++ + 933 +++ + 934 +++ + 935 ++ + 936 +++ + 937 +++ + 938 +++ + 939 +++ + 940 +++ + 941 +++ + 942 +++ + 943 ++ + 944 +++ + 945 +++ + 946 +++ + 947 +++ + 948 +++ + 949 +++ + 950 +++ + 951 +++ + 952 +++ + 953 +++ + 954 +++ + 955 +++ + 956 +++ + 957 +++ + 958 +++ + 959 ++ + 960 +++ + 961 +++ + 962 +++ + 963 +++ + 964 +++ + 965 ++ + 966 +++ + 967 +++ + 968 +++ + 969 ++ + 970 +++ + 971 +++ + 972 +++ + 973 ++ + 974 ++ + 975 ++ + 976 +++ + 977 +++ + 978 +++ + 979 +++ + 980 +++ + 981 +++ ++ 982 +++ + 983 +++ + 984 +++ + 985 +++ + 986 +++ + 987 +++ + 988 +++ + 989 +++ + 990 +++ + 991 +++ + 992 ++ + 993 +++ + 994 +++ + 995 +++ + 996 +++ + 997 +++ + 998 +++ + 999 +++ + 1000 ++ + 1001 +++ + 1002 +++ + 1003 +++ + 1004 +++ + 1005 +++ + 1006 +++ + 1007 +++ + 1008 +++ + 1009 +++ + 1010 +++ + 1011 +++ + 1012 +++ + 1013 +++ + 1014 +++ + 1015 +++ + 1016 +++ + 1017 +++ + 1018 +++ + 1019 +++ + 1020 +++ + 1021 +++ + 1022 +++ + 1023 +++ + 1024 +++ + 1025 + + 1026 ++ + 1027 +++ + 1028 ++ + 1029 +++ + 1030 ++ + 1031 +++ + 1032 +++ + 1033 +++ + 1034 +++ + 1035 +++ + 1036 +++ + 1037 ++ + 1038 +++ + 1039 +++ + 1040 +++ + 1041 +++ + 1042 ++ + 1043 +++ + 1044 ++ + 1045 +++ + 1046 +++ + 1047 +++ + 1048 +++ + 1049 +++ + 1050 +++ + 1051 +++ + 1052 ++ + 1053 ++ + 1054 ++ + 1055 ++ + 1056 ++ + 1057 +++ + 1058 +++ + 1059 +++ + 1060 +++ + 1061 +++ + 1062 +++ + 1063 +++ + 1064 +++ + 1065 +++ + 1066 ++ + 1067 +++ + 1068 + + 1069 ++ + 1070 +++ + 1071 +++ + 1072 +++ + 1073 +++ + 1074 +++ + 1075 +++ + 1076 +++ + 1077 +++ + 1078 +++ + 1079 +++ + 1080 +++ + 1081 +++ + 1082 +++ + 1083 +++ + 1084 +++ + 1085 +++ + 1086 +++ + 1087 +++ + 1088 +++ + 1089 +++ + 1090 +++ + 1091 +++ + 1092 +++ + 1093 +++ + 1094 +++ + 1095 +++ + 1096 ++ + 1097 ++ + 1098 ++ + 1099 +++ + 1100 ++ + 1101 +++ + 1102 +++ + 1103 +++ + 1104 +++ + 1105 +++ + 1106 +++ + 1107 +++ + 1108 +++ + 1109 +++ + 1110 +++ + 1111 ++ + 1112 ++ + 1113 ++ + 1114 +++ + 1115 +++ + 1116 +++ + 1117 ++ + 1118 +++ + 1119 +++ + 1120 ++ + 1121 +++ + 1122 +++ + 1123 ++ + 1124 ++ + 1125 +++ + 1126 +++ + 1127 +++ + 1128 +++ + 1129 +++ + 1130 ++ + 1131 ++ + 1132 +++ + 1133 +++ + 1134 +++ + 1135 +++ + 1136 +++ + 1137 +++ + 1138 +++ + 1139 +++ + 1140 +++ + 1141 +++ + 1142 +++ + 1143 +++ + 1144 +++ + 1145 +++ + 1146 +++ + 1147 +++ + 1148 +++ + 1149 +++ + 1150 +++ + 1151 +++ + 1152 +++ + 1153 +++ + 1154 +++ + 1155 +++ + 1156 +++ + 1157 ++ + 1158 +++ + 1159 +++ + 1160 +++ ++ 1161 +++ ++ 1162 +++ ++ 1163 +++ ++ 1164 +++ + 1165 +++ ++ 1166 +++ + 1167 +++ ++ 1168 +++ + 1169 +++ + 1170 +++ + 1171 +++ + 1172 +++ + 1173 +++ + 1174 +++ + 1175 +++ + 1176 +++ + 1177 +++ + 1178 +++ + 1179 +++ + 1180 +++ + 1181 +++ + 1182 +++ + 1183 +++ + 1184 +++ + 1185 +++ + 1186 +++ + 1187 +++ + 1188 +++ + 1189 +++ + 1190 +++ + 1191 +++ + 1192 +++ + 1193 +++ + 1194 +++ + 1195 +++ + 1196 +++ + 1197 +++ + 1198 +++ + 1199 +++ + 1200 +++ + 1201 +++ + 1202 +++ + 1203 ++ + 1204 +++ + 1205 +++ + 1206 +++ + 1207 +++ + 1208 +++ + 1209 +++ + 1210 +++ + 1211 ++ + 1212 +++ + 1213 +++ + 1214 +++ + 1215 +++ + 1216 +++ + 1217 ++ + 1218 +++ + 1219 +++ + 1220 +++ + 1221 +++ + 1222 +++ + 1223 +++ + 1224 +++ + 1225 +++ + 1226 +++ + 1227 +++ + 1228 +++ + 1229 +++ + 1230 +++ + 1231 +++ + 1232 +++ +

Example 22

Human CDK6/Cyclin D3 Inhibitory Activity

CDK6/cyclin D3 inhibitory activity was determined by the off-chip mobility shift assay (MSA). The MSA separates proteins from one another on the basis of a difference in electrophoretic mobility depending on the molecular weight or electric charge of the proteins. The kinase activity is determined by quantifying the degree of phosphorylation through electrophoretic analysis of a positive to negative change in electric charge of the substrate phosphorylated by the kinase.

Each solution was prepared with an assay buffer containing 20 mM HEPES (pH 7.5), 0.01% Triton X-100, and 2 mM dithiothreitol. A test compound solution was prepared by dilution of the test compound with dimethyl sulfoxide (DMSO) to a concentration 100 times higher than the final concentration and then 25-fold dilution with the assay buffer to a concentration four times higher than the final concentration. An ATP/substrate/metal solution was prepared to have a concentration four times higher than the final concentration. An enzyme solution was prepared to have a concentration twice higher than the final concentration. The final enzyme concentration was adjusted to an appropriate level on the basis of the enzyme activity signal and the inhibitory activity of a positive control compound.

The test compound solution (5 μL/well) and the ATP/substrate/metal solution (5 μL/well) were added to a 384-well plate, and the enzyme solution or the assay buffer (10 μL/well) was added to the plate (total amount of the reaction mixture: 20 μL/well) for initiation of enzymatic reaction. The reaction mixture had a composition of 20 mM HEPES (pH 7.5), 0.01% Triton X-100, 2 mM dithiothreitol, 1000 nM peptide substrate (DYRKtide-F), 300 μM ATP, 5 mM magnesium chloride, 1% DMSO, and a predetermined concentration of CDK6/cyclin D3. The reaction was performed at room temperature for five hours, and a termination buffer (QuickScout Screening Assist MSA, manufactured by Carna Biosciences, Inc.) (60 μL/well) was then added to the plate for termination of the reaction. Subsequently, the substrate peptide and the phosphorylated peptide in the reaction mixture were separated from each other and quantified with LabChip 3000 (manufactured by Caliper Lifesciences). The kinase reaction was evaluated by the product ratio (P/(P+S)) calculated from the peak height (S) of the substrate peptide and the peak height (P) of the phosphorylated peptide.

The percent inhibition of enzyme activity was calculated for each test compound (note: enzyme activity=100% in the case of addition of the enzyme solution and addition of DMSO instead of the test compound solution, whereas enzyme activity=0% in the case of addition of the assay buffer instead of the enzyme solution and addition of DMSO instead of the test compound solution). The percent inhibition of enzyme activity was fitted to a dose-response curve, to determine a 50% inhibitory concentration against CDK6/cyclin D3.

The inhibitory activity of each compound against CDK6/cyclin D3 was shown in tables described below. In each table, “+++” corresponds to IC₅₀<10 nM, “++” 10 nM≤IC₅₀<100 nM, and “+” 100 nM≤IC₅₀.

TABLE 7 CDK6 CDK6 Compound Activity Compound Activity No. Symbol No. Symbol 3 +++ 16 +++ 217 ++ 220 ++ 221 ++ 230 +++ 300 ++ 304 ++ 363 ++ 672 ++ 749 ++ 778 ++ 779 +++ 818 ++ 819 ++ 843 ++ 844 +++ 879 +++ 883 +++ 904 +++ 929 +++ 938 ++ 941 +++ 954 +++ 1036 ++

Example 23

A monoclonal antibody cocktail against type II collagen (Arthritogenic MoAb Cocktail (Chondrex #53100), 4.8 mg/mL) was intraperitoneally administered (250 μL/head) to a group of mice with collagen antibody-induced arthritis (CAIA) (vehicle/+, group of drug administration) (day 1). LPS (LPS Solution (E. coli 0111:B4) (Chondrex #9028), 0.5 mg/mL) was intraperitoneally administered (100 μL/head) on day 4, to induce the disease. The drug was evaluated on the basis of pathological scoring until day 9. The drug was orally administered consecutively from day 4 to day 8 once a day.

In mice of a non-disease-induced group (vehicle/-group), PBS (pH 7.2, Gibco #20012-027) was intraperitoneally administered (250 μL/head) on day 1, and LPS was intraperitoneally administered on day 4.

The drug was evaluated on the basis of the pathological scoring (score 0 to score 4 for each of the extremities, evaluated by the total score). Scoring criteria are as follows:

score 0: no change;

score 1: swelling of only one limb;

score 2: swelling of wrist and ankle or swelling of two or more limbs;

score 3: swelling of wrist and ankle and swelling of one or more limbs; and

score 4: swelling of wrist and ankle and swelling of all the limbs.

FIG. 1 shows the results (scores) on day 9 (final day).

LENGTHY TABLES The patent contains a lengthy table section. A copy of the table is available in electronic form from the USPTO web site (http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US10124004B2). An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3). 

The invention claimed is:
 1. A compound represented by Formula (I) or a pharmaceutically acceptable salt thereof:

[where L represents —NR⁵—, —O—, or —S—; R⁵ represents a hydrogen atom or a C₁₋₆ alkyl group substituted with zero to two —OH groups, zero to two C₁₋₈ alkoxy groups, and zero to six fluorine atoms; R¹ represents a C₁₋₈ alkyl, C₃₋₁₂ cycloalkyl, (C₃₋₁₂ cycloalkyl)-C₁₋₆ alkyl, 4- to 12-membered heterocyclyl, (4- to 12-membered heterocyclyl)-C₁₋₆ alkyl, C₆₋₁₀ aryl, (C₆₋₁₀ aryl)-C₁₋₆ alkyl, 5- to 10-membered heteroaryl, (5- to 10-membered heteroaryl)-C₁₋₆ alkyl, C₁₋₈ alkylsulfonyl, or C₁₋₈ acyl group; each of the heteroatom-containing groups represented by R¹ contains one to four heteroatoms selected from oxygen, sulfur, and nitrogen atoms; R¹ is optionally substituted with one to six substituents selected from the group consisting of a halogen atom, ═O, —OH, —CN, —COOH, —COOR⁶, —R⁷, a C₃₋₆ cycloalkyl group substituted with zero to two —OH groups, zero to two C₁₋₈ alkoxy groups, and zero to six fluorine atoms, a 3- to 10-membered heterocyclyl group substituted with zero to two —OH groups, zero to two C₁₋₈ alkoxy groups, and zero to six fluorine atoms, a C₁₋₈ acyl group substituted with zero to two —OH groups, zero to two C₁₋₈ alkoxy groups, and zero to six fluorine atoms, and a C₁₋₈ alkoxy group substituted with zero to two —OH groups, zero to two C₁₋₈ alkoxy groups, and zero to six fluorine atoms; R⁶ and R⁷ each independently represent a C₁₋₆ alkyl group substituted with zero to two —OH groups, zero to two C₁₋₈ alkoxy groups, and zero to six fluorine atoms; R² represents a C₁₋₈ alkyl, C₃₋₈ cycloalkyl, 4- to 6-membered heterocyclyl, or C₁₋₈ acyl group, —COOR⁸, or —CONR⁹R¹⁰; each of the C₁₋₈ alkyl and C₃₋₈ cycloalkyl groups represented by R² is substituted with zero or one —OH group, zero to two C₁₋₈ alkoxy groups substituted with zero or one —OH group, zero or one C₁₋₄ alkoxy group, and zero to three fluorine atoms, and zero to five fluorine atoms; R² is neither an unsubstituted C₁₋₈ alkyl, nor unsubstituted C₃₋₈ cycloalkyl, nor trifluoromethyl group; R⁸, R⁹, and R¹⁰ each independently represent a hydrogen atom or a C₁₋₈ alkyl group; the 4- to 6-membered heterocyclyl group represented by R² is optionally substituted with one to four substituents selected from the group consisting of a fluorine atom, —OH, and C₁₋₄ alkyl and C₁₋₄ alkoxy groups; each of the C₁₋₈ acyl group, —COOR⁸, and —CONR⁹R¹⁰ represented by R² is optionally substituted with one to four substituents selected from the group consisting of a fluorine atom, —OH, and a C₁₋₄ alkoxy group; R⁹ and R¹⁰ of —CONR⁹R¹⁰ represented by R² are optionally bonded via a single bond or —O— to form a ring including the nitrogen atom bonded to R⁹ and R¹⁰; the heterocyclyl group represented by R² having a 4- or 5-membered ring contains one oxygen heteroatom, and the heterocyclyl group having a 6-membered ring contains one or two oxygen heteroatoms; R³ represents a hydrogen atom, a C₁₋₈ alkyl group, or a halogen atom; X represents CR¹¹ or a nitrogen atom; Y represents CR¹² or a nitrogen atom; Z represents CR¹³ or a nitrogen atom; R¹¹ to R¹³ each independently represent a hydrogen, fluorine, or chlorine atom or a C₁₋₆ alkyl or C₁₋₆ alkoxy group; R⁴ represents -A¹-A²-A³; A¹ represents a single bond or a C₁₋₈ alkylene, C₂₋₈ alkenylene, or C₂₋₈ alkynylene group; one or two sp³ carbon atoms at any positions of A¹ are optionally replaced with one or two structures selected from the group consisting of —O—, —NR¹⁴—, —C(═O)—, —C(═O)—O—, —O—C(═O)—, —O—C(═O)—O—, —C(═O)—NR⁵—, —O—C(═O)—NR¹⁶—, —NR¹⁷—C(═O)—, —NR¹⁸—C(═O)—O—, —NR¹⁹—C(═O)—NR²⁰—, —S(═O)_(p)—, —S(═O)₂—NR²¹—, —NR²²—S(═O)₂—, and —NR²³—S(═O)₂—NR²⁴—, and a structure of —O—O—, —O—NR¹⁴—, —NR¹⁴—O—, —O—CH₂—O—, —O—CH₂—NR¹⁴—, or —NR¹⁴—CH₂—O— is not formed in the case of replacement of two sp³ carbon atoms; A² represents a single bond or a C₁₋₇ alkylene, C₃₋₁₂ cycloalkylene, C₃₋₁₂ cycloalkylidene, 4- to 12-membered heterocyclylene, 4- to 12-membered heterocyclylidene, C₆₋₁₀ arylene, or 5- to 10-membered heteroarylene group; A³ represents a halogen atom, —CN, —NO₂, —R²⁵, —OR²⁶, —NR²⁷R²⁸, —C(═O)R²⁹, —C(═O)—OR³⁰, —O—C(═O)R³¹, —O—C(═O)—NR³²R³³, —C(═O)—NR³⁴R³⁵, —NR³⁶—C(═O)R³⁷, —NR³⁸—C(═O)—OR³⁹, —S(═O)₂—R⁴⁰, —S(═O)₂—NR⁴¹R⁴², or —NR⁴³—S(═O)₂R⁴⁴; A³ represents —R²⁵, if the A¹ end on the A² side has a structure selected from the group consisting of —O—, —NR¹⁴—, —C(═O)—, —C(═O)—O—, —O—C(═O)—, —O—C(═O)—O—, —C(═O)—NR¹⁵—, —O—C(═O)—NR¹⁶—, —NR¹⁷—C(═O)—, —NR¹⁸—C(═O)—O—, —NR¹⁹—C(═O)—NR²⁰—, —S(═O)_(p)—, —S(═O)₂—NR²¹—, —NR²²—S(═O)₂—, and —NR²³—S(═O)₂—NR²⁴— and A² is a single bond; R¹⁴, R³², R³⁴, R³⁶, R³⁸, R⁴¹, and R⁴³ each independently represent a hydrogen atom or a C₁₋₈ alkyl, C₁₋₈ acyl, C₁₋₈ alkylsulfonyl, 4- to 12-membered heterocyclyl, C₃₋₁₂ cycloalkyl, C₆₋₁₀ aryl, 5- to 10-membered heteroaryl, (4- to 12-membered heterocyclyl)-C₁₋₃ alkyl, (C₃₋₁₂ cycloalkyl)-C₁₋₃ alkyl, (C₆₋₁₀ aryl)-C₁₋₃ alkyl, or (5- to 10-membered heteroaryl)-C₁₋₃ alkyl group; R¹⁵ to R³¹, R³³, R³⁵, R³⁷, R³⁹, R⁴⁰, R⁴², and R⁴⁴ each independently represent a hydrogen atom or a C₁₋₈ alkyl, 4- to 12-membered heterocyclyl, C₃₋₁₂ cycloalkyl, C₆₋₁₀ aryl, 5- to 10-membered heteroaryl, (4- to 12-membered heterocyclyl)-C₁₋₃ alkyl, (C₃₋₁₂ cycloalkyl)-C₁₋₃ alkyl, (C₆₋₁₀ aryl)-C₁₋₃ alkyl, or (5- to 10-membered heteroaryl)-C₁₋₃ alkyl group; A¹, A², A³, and R¹⁴ to R⁴⁴ in A¹, A², and A³ are each optionally substituted with one to four substituents selected from the group consisting of —OH, ═O, —COOH, —SO₃H, —PO₃H, —CN, —NO₂, a halogen atom, a C₁₋₈ alkyl group substituted with zero to two —OH groups, zero to two —OR⁴⁵ groups, and zero to six fluorine atoms, a C₃₋₁₂ cycloalkyl group substituted with zero to two —OH groups, zero to two —OR⁴⁶ groups, and zero to six fluorine atoms, a C₁₋₈ alkoxy group substituted with zero to two —OH groups, zero to two —OR⁴⁷ groups, and zero to six fluorine atoms, and a 4- to 12-membered heterocyclyl group substituted with zero to two —OH groups, zero to two —OR⁴⁹ groups, and zero to six fluorine atoms; R¹⁴ to R⁴⁴ are optionally bonded in A¹, A², or A³ or between A¹ and A², between A¹ and A³, or between A² and A³ via a single bond, —O—, —NR⁵⁰—, or —S(═O)_(p)— to form a ring; R¹¹ or R¹³ is optionally bonded to A¹, A², or A³ via a single bond, —O—, —NR⁵¹—, or —S(═O)_(p)— to form a ring; R⁴⁵ to R⁵¹ each represent a hydrogen atom or a C₁₋₄ alkyl group substituted with zero or one —OH group and zero to six fluorine atoms; p represents an integer of 0 to 2; and each of the heteroatom-containing groups represented by A¹, A², and A³ contains one to four heteroatoms selected from oxygen, sulfur, and nitrogen atoms].
 2. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein L represents —NH—.
 3. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R¹ represents a C₁₋₈ alkyl, C₃₋₁₂ cycloalkyl, (C₃₋₁₂ cycloalkyl)-C₁₋₆ alkyl, 4- to 12-membered heterocyclyl, or (4- to 12-membered heterocyclyl)-C₁₋₆ alkyl group.
 4. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R² is a C₁₋₈ alkyl group substituted with one to four fluorine atoms.
 5. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R² is a C₁₋₈ alkyl group substituted with zero or one —OH group and zero to two C₁₋₈ alkoxy groups substituted with zero or one —OH group, zero or one C₁₋₄ alkoxy group, and zero to three fluorine atoms.
 6. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R² is a 4- to 6-membered heterocyclyl group optionally substituted with one to four substituents selected from the group consisting of a fluorine atom, —OH, and C₁₋₄ alkyl and C₁₋₄ alkoxy groups.
 7. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R² is —COOR⁸, —CONR⁹R¹⁰, or a C₁₋₈ acyl group, optionally each group being substituted with one to four substituents selected from the group consisting of a fluorine atom, —OH, and a C₁₋₈ alkoxy group.
 8. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein X represents CR¹¹, Y represents CR¹², and Z represents CR¹³.
 9. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein X represents a nitrogen atom, Y represents CR¹², and Z represents CR¹³.
 10. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein X represents CR¹¹, Y represents a nitrogen atom, and Z represents CR¹³.
 11. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein X represents CR¹¹, Y represents CR¹², and Z represents a nitrogen atom.
 12. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein A¹ is a single bond.
 13. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein A¹ represents a C₁₋₈ alkylene group, and no sp³ carbon atom in A¹ is replaced with another structure.
 14. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein A¹ represents a C₁₋₈ alkylene group, and one sp³ carbon atom at any position of A¹ is replaced with —O—.
 15. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein A¹ represents a C₁₋₈ alkylene group, and one sp³ carbon atom at any position of A¹ is replaced with —NR¹⁴—.
 16. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein A¹ represents a C₁₋₈ alkylene group, one sp³ carbon atom at any position of A¹ is replaced with —NR¹⁴—, and one sp³ carbon atom at any other position of A¹ is optionally replaced with —O—.
 17. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein A² represents a 4- to 12-membered heterocyclylene group; and A² is optionally substituted with one to four substituents selected from the group consisting of —OH, —COOH, —SO₃H, —PO₃H, —CN, —NO₂, a halogen atom, a C₁₋₈ alkyl group substituted with zero to two —OH groups, zero to two —OR⁴⁵ groups, and zero to six fluorine atoms, a C₃₋₁₂ cycloalkyl group substituted with zero to two —OH groups, zero to two —OR⁴⁶ groups, and zero to six fluorine atoms, a C₁₋₈ alkoxy group substituted with zero to two —OH groups, zero to two —OR⁴⁷ groups, and zero to six fluorine atoms, and a 4- to 12-membered heterocyclyl group substituted with zero to two —OH groups, zero to two —OR⁴⁹ groups, and zero to six fluorine atoms.
 18. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein A² represents a 4- to 12-membered heterocyclylene group substituted with ═O; and A² is optionally substituted with one to four substituents selected from the group consisting of —OH, ═O, —COOH, —SO₃H, —PO₃H, —CN, —NO₂, a halogen atom, a C₁₋₈ alkyl group substituted with zero to two —OH groups, zero to two —OR⁴⁵ groups, and zero to six fluorine atoms, a C₃₋₁₂ cycloalkyl group substituted with zero to two —OH groups, zero to two —OR⁴⁶ groups, and zero to six fluorine atoms, a C₁₋₈ alkoxy group substituted with zero to two —OH groups, zero to two —OR⁴⁷ groups, and zero to six fluorine atoms, and a 4- to 12-membered heterocyclyl group substituted with zero to two —OH groups, zero to two —OR⁴⁹ groups, and zero to six fluorine atoms.
 19. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein X represents CR¹¹, Y represents CR¹², Z represents CR¹³, and R¹¹ or R¹³ is bonded to A¹, A², or A³ via a single bond, —O—, —NR⁵¹—, or —S(═O)_(p)— to form a ring.
 20. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein A³ is a hydrogen atom.
 21. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein A³ is a halogen atom, —CN, —R²⁵, —OR²⁶, —NR²⁷R²⁸, —C(═O)R²⁹, or —C(═O)—OR³⁰, and R²⁵ to R³⁰ each independently represent a hydrogen atom, an optionally substituted C₁₋₈ alkyl group, an optionally substituted 4- to 12-membered heterocyclyl group, an optionally substituted C₃₋₁₂ cycloalkyl group, an optionally substituted (4- to 12-membered heterocyclyl)-C₁₋₃ alkyl group, or an optionally substituted (C₃₋₁₂ cycloalkyl)-C₁₋₃ alkyl group.
 22. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R³ is a hydrogen atom.
 23. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R³ represents a C₁₋₄ alkyl group, a fluorine atom, or a chlorine atom.
 24. The compound, or pharmaceutically acceptable salt thereof, selected from; 6-(difluoromethyl)-N8-isopropyl-N2-(5-piperazin-1-yl-2-pyridyl)pyrido[3,4-d]pyrimidine-2,8-diamine (1R)-1-[8-(isopropylamino)-2-[(5-piperazin-1-yl-2-pyridyl)amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol 1-[2-[(5-piperazin-1-yl-2-pyridyl)amino]-8-(tetrahydrofuran-3-ylamino)pyrido[3,4-d]pyrimidin-6-yl]ethanol 1-[2-[(5-piperazin-1-yl-2-pyridyl)amino]-8-(tetrahydropyran-3-ylamino)pyrido[3,4-d]pyrimidin-6-yl]ethanol N8-isopropyl-6-[(1R)-1-methoxyethyl]-N2-(6-piperazin-1-ylpyridazin-3-yl)pyrido[3,4-d]pyrimidine-2,8-diamine N8-isopropyl-6-[(1R)-1-methoxyethyl]-N2-[5-(piperazin-1-ylmethyl)-2-pyridyl]pyrido[3,4-d]pyrimidine-2,8-diamine 1-[6-[[6-[(1R)-1-hydroxyethyl]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]piperazin-2-one 1-[6-[[5-chloro-6-[(1R)-1-hydroxyethyl]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]piperazin-2-one (1R)-1-[2-[(6-piperazin-1-ylpyridazin-3-yl)amino]-8-(tetrahydropyran-4-ylamino)pyrido[3,4-d]pyrimidin-6-yl]ethanol (1R)-1-[2-[(6-piperazin-1-ylpyridazin-3-yl)amino]-8-[[(3S)-tetrahydropyran-3-yl]amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol (1R)-1-[2-[(6-piperazin-1-ylpyridazin-3-yl)amino]-8-[[(3R)-tetrahydropyran-3-yl]amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol (1R)-1-[2-[[5-(piperazin-1-ylmethyl)-2-pyridyl]amino]-8-(tetrahydropyran-4-ylamino)pyrido[3,4-d]pyrimidin-6-yl]ethanol (1R)-1-[2-[[5-(piperazin-1-ylmethyl)-2-pyridyl]amino]-8-[[(3S)-tetrahydropyran-3-yl]amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol (1R)-1-[2-[[5-(piperazin-1-ylmethyl)-2-pyridyl]amino]-8-[[(3R)-tetrahydropyran-3-yl]amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol 1-[6-[[6-[(1R)-1-hydroxyethyl]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-2-yl]amino]pyridazin-3-yl]piperidin-4-ol (1R)-1-[8-(isopropylamino)-2-[(6-piperazin-1-ylpyridazin-3-yl)amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol 1-[[6-[[6-[(1R)-1-hydroxyethyl]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]methyl]piperazin-2-one 6-[(1R)-1-methoxyethyl]-N2-[5-(piperazin-1-ylmethyl)-2-pyridyl]-N8-[(3S)-tetrahydropyran-3-yl]pyrido[3,4-d]pyrimidine-2,8-diamine 6-[(1R)-1-methoxyethyl]-N2-(6-piperazin-1-ylpyridazin-3-yl)-N8-[(3S)-tetrahydropyran-3-yl]pyrido[3,4-d]pyrimidine-2,8-diamine 6-[(1R)-1-methoxyethyl]-N2-[5-(piperazin-1-ylmethyl)-2-pyridyl]-N8-(tetrahydropyran-4-ylmethyl)pyrido[3,4-d]pyrimidine-2,8-diamine N8-isopropyl-6-[(1R)-1-methoxyethyl]-N2-(5-piperazin-1-ylpyrazin-2-yl)pyrido[3,4-d]pyrimidine-2,8-diamine N8-isopropyl-6-[(1R)-1-methoxyethyl]-N2-[6-[(2S)-2-methylpiperazin-1-yl]pyridazin-3-yl]pyrido[3,4-d]pyrimidine-2,8-diamine N8-isopropyl-6-[(1R)-1-methoxyethyl]-N2-[6-[(2R)-2-methylpiperazin-1-yl]pyridazin-3-yl]pyrido[3,4-d]pyrimidine-2,8-diamine (1R)-1-[2-[[6-(4,7-diazaspiro[2.5]octan-7-yl)pyridazin-3-yl]amino]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-6-yl]ethanol (1R)-1-[2-[[5-(4,7-diazaspiro[2.5]octan-7-ylmethyl)-2-pyridyl]amino]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-6-yl]ethanol 2-[1-[[6-[[6-[(1R)-1-hydroxyethyl]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]methyl]-4-piperidyl]propan-2-ol (1R)-1-[2-[[5-[[4-(2-hydroxyethyl)piperazin-1-yl]methyl]-2-pyridyl]amino]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-6-yl]ethanol (1R)-1-[2-[[5-[2-(dimethylamino)ethoxy]-2-pyridyl]amino]-8-[[(3S)-tetrahydropyran-3-yl]amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol (1R)-1-[2-[[6-(4-methylpiperazin-1-yl)pyridazin-3-yl]amino]-8-[[(3S)-tetrahydropyran-3-yl]amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol 2-hydroxy-1-[4-[6-[[6-[(1R)-1-hydroxyethyl]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-2-yl]amino]pyridazin-3-yl]piperazin-1-yl]ethanone 1-[6-[[8-(isopropylamino)-6-[(2S)-tetrahydrofuran-2-yl]pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]piperazin-2-one (1R)-1-[8-(isopropylamino)-2-(5,6,7,8-tetrahydro-1,6-naphthyridin-2-ylamino)pyrido[3,4-d]pyrimidin-6-yl]ethanol 2-[4-[[6-[[6-[(1R)-1-hydroxyethyl]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]methyl]piperazin-1-yl]-2-methyl-propan-1-ol 4-[6-[[6-[(1R)-1-hydroxyethyl]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]-1-[(2S)-2-hydroxypropyl]-1,4-diazepan-5-one 4-[6-[[6-[(1R)-1-hydroxyethyl]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]-1-[(2R)-2-hydroxypropyl]-1,4-diazepan-5-one N8-isopropyl-N2-[5-(piperazin-1-ylmethyl)-2-pyridyl]-6-[(2S)-tetrahydrofuran-2-yl]pyrido[3,4-d]pyrimidine-2,8-diamine 1-[6-[[6-[(1R)-1-hydroxyethyl]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-2-yl]amino]-2-methyl-3-pyridyl]piperazin-2-one 1-[6-[[8-(isopropylamino)-6-[(3S)-tetrahydrofuran-3-yl]pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]piperazin-2-one (1R)-1-[2-(5,6,7,8-tetrahydro-1,6-naphthyridin-2-ylamino)-8-[[(3S)-tetrahydropyran-3-yl]amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol 1-[6-[[8-(isopropylamino)-6-(3-methyloxetan-3-yl)pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]piperazin-2-one (1R)-1-[2-[[5-[4-(dimethylamino)cyclohexoxy]-2-pyridyl]amino]-8-[[(3S)-tetrahydropyran-3-yl]amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol 6-[(1R)-1-methoxyethyl]-N2-[5-(piperazin-1-ylmethyl)-2-pyridyl]-N8-propyl-pyrido[3,4-d]pyrimidine-2,8-diamine 6-[(1R)-1-methoxyethyl]-N2-(6-piperazin-1-ylpyridazin-3-yl)-N8-propyl-pyrido[3,4-d]pyrimidine-2,8-diamine 1-[[6-[[6-(difluoromethyl)-8-[(4-methylcyclohexyl)amino]pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]methyl]piperidine-4-carboxylic acid (1R)-1-[8-(ethylamino)-2-[[5-[[4-(2-hydroxyethyl)piperazin-1-yl]methyl]-2-pyridyl]amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol (1R)-1-[2-[[5-[[4-(2-hydroxyethyl)piperazin-1-yl]methyl]-2-pyridyl]amino]-8-(propylamino)pyrido[3,4-d]pyrimidin-6-yl]ethanol N8-isopropyl-6-(3-methyloxetan-3-yl)-N2-(6-piperazin-1-ylpyridazin-3-yl)pyrido[3,4-d]pyrimidine-2,8-diamine N8-isopropyl-6-(3-methyloxetan-3-yl)-N2-[5-(piperazin-1-ylmethyl)-2-pyridyl]pyrido[3,4-d]pyrimidine-2,8-diamine 6-(3-methyloxetan-3-yl)-N2-[5-(piperazin-1-ylmethyl)-2-pyridyl]-N8-[(3S)-tetrahydropyran-3-yl]pyrido[3,4-d]pyrimidine-2,8-diamine 4-[6-[[6-[(1R)-1-hydroxyethyl]-8-[isopropyl(methyl)amino]pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]-1,4-diazepan-5-one (1R)-1-[8-(isopropylamino)-2-[(6-methyl-5-piperazin-1-yl-2-pyridyl)amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol (1R)-1-[2-[[6-(2-hydroxyethyl)-7,8-dihydro-5H-1,6-naphthyridin-2-yl]amino]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-6-yl]ethanol (1R)-1-[8-(isopropylamino)-2-[[6-[2-(methylamino)ethyl]-7,8-dihydro-5H-1,6-naphthyridin-2-yl]amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol N2-(6-piperazin-1-ylpyridazin-3-yl)-6-[(3S)-tetrahydrofuran-3-yl]-N8-[(3S)-tetrahydropyran-3-yl]pyrido[3,4-d]pyrimidine-2,8-diamine N2-[5-(piperazin-1-ylmethyl)-2-pyridyl]-6-[(3R)-tetrahydrofuran-3-yl]-N8-[(3S)-tetrahydropyran-3-yl]pyrido[3,4-d]pyrimidine-2,8-diamine (1R)-1-[2-[[6-[2-(dimethylamino)ethyl]-7,8-dihydro-5H-1,6-naphthyridin-2-yl]amino]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-6-yl]ethanol (2S)-1-[4-[[6-[[8-(ethylamino)-6-[(1R)-1-hydroxyethyl]pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]methyl]piperazin-1-yl]propan-2-ol (2R)-1-[4-[[6-[[8-(ethylamino)-6-[(1R)-1-hydroxyethyl]pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]methyl]piperazin-1-yl]propan-2-ol (1R)-1-[8-(isopropylamino)-2-[[5-[(2R)-2-methylpiperazin-1-yl]-2-pyridyl]amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol (1R)-1-[8-(isopropylamino)-2-[[5-[(2S)-2-methylpiperazin-1-yl]-2-pyridyl]amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol N8-isopropyl-N2-(5-piperazin-1-yl-2-pyridyl)-6-[(2S)-tetrahydrofuran-2-yl]pyrido[3,4-d]pyrimidine-2,8-diamine (1R)-1-[8-(cyclobutylamino)-2-[[5-[[4-(2-hydroxyethyl)piperazin-1-yl]methyl]-2-pyridyl]amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol (1R)-1-[8-(cyclopropylmethylamino)-2-[[5-[[4-(2-hydroxyethyl)piperazin-1-yl]methyl]-2-pyridyl]amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol 6-(3-methyloxetan-3-yl)-N2-(5-piperazin-1-yl-2-pyridyl)-N8-propyl-pyrido[3,4-d]pyrimidine-2,8-diamine 6-(3-methyloxetan-3-yl)-N2-[5-(piperazin-1-ylmethyl)-2-pyridyl]-N8-propyl-pyrido[3,4-d]pyrimidine-2,8-diamine N2-(5-piperazin-1-yl-2-pyridyl)-N8-propyl-6-tetrahydrofuran-3-yl-pyrido[3,4-d]pyrimidine-2,8-diamine N2-[5-(piperazin-1-ylmethyl)-2-pyridyl]-N8-propyl-6-tetrahydrofuran-3-yl-pyrido[3,4-d]pyrimidine-2,8-diamine N8-isopropyl-6-(3-methyloxetan-3-yl)-N2-(5-piperazin-1-yl-2-pyridyl)pyrido[3,4-d]pyrimidine-2,8-diamine N8-isopropyl-N2-(5-piperazin-1-yl-2-pyridyl)-6-tetrahydrofuran-3-yl-pyrido[3,4-d]pyrimidine-2,8-diamine 2-[4-[[6-[[8-(isopropylamino)-6-tetrahydrofuran-3-yl-pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]methyl]piperazin-1-yl]ethanol 2-[4-[[6-[[6-tetrahydrofuran-3-yl-8-[[(3S)-tetrahydropyran-3-yl]amino]pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]methyl]piperazin-1-yl]ethanol (1R)-1-[2-[[5-[[4-(hydroxymethyl)-1-piperidyl]methyl]-2-pyridyl]amino]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-6-yl]ethanol 1-[[6-[[6-[(1R)-1-hydroxyethyl]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]methyl]piperidin-4-ol 1-[[6-[[8-(tert-butylamino)-6-[(1R)-1-hydroxyethyl]pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]methyl]piperidin-4-ol (1R)-1-[8-(tert-butylamino)-2-[[5-[[4-(hydroxymethyl)-1-piperidyl]methyl]-2-pyridyl]amino]pyrido[3,4-d]pyrimidin-6-yl]ethanol 1-[[6-[[6-[(1R)-1-hydroxyethyl]-8-(isobutylamino)pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]methyl]piperidin-4-ol (1R)-1-[2-[[5-[[4-(hydroxymethyl)-1-piperidyl]methyl]-2-pyridyl]amino]-8-(isobutylamino)pyrido[3,4-d]pyrimidin-6-yl]ethanol 1-[6-[[6-[(1R)-1-hydroxypropyl]-8-(isopropylamino)pyrido[3,4-d]pyrimidin-2-yl]amino]-3-pyridyl]piperazin-2-one (1R)-1-[2-[[5-[[4-(2-hydroxyethyl)piperazin-1-yl]methyl]-6-methyl-2-pyridyl]amino]-8-(propylamino)pyrido[3,4-d]pyrimidin-6-yl]ethanol.
 25. A pharmaceutical composition comprising the compound or pharmaceutically acceptable salt thereof according to claim 1 and a pharmaceutically acceptable carrier.
 26. A pharmaceutical composition exhibiting a CDK4/6 inhibitory activity, comprising the compound or pharmaceutically acceptable salt thereof according to claim 1 as an active ingredient.
 27. A drug for prevention or treatment of rheumatoid arthritis, arteriosclerosis, pulmonary fibrosis, cerebral infarction, or cancer, the drug comprising the compound or pharmaceutically acceptable salt thereof according to claim 1 as an active ingredient. 